mproving p450 bm3 towards conversion

IMPROVING P450 BM3 TOWARDS CONVERSION OF QUATERNARY

AMINES Using simultaneous site directed mutagenesis in generating mutant libraries of P450 BM3 for screening to yield the best performing mutant that can be applicable in further processes together in gaining in depth knowledge at the catalytic efficiency of cytochrome P450 BM3.

Bachelor Thesis

NOVEMBER 23, 2016 RWTH AACHEN UNIVERSITY INSTITUTE FOR BIOTECHNOLOGY

Worringerweg 3, 52074 Aachen, Germany

Author : Merve Keser

1 IMPROVING P450 BM3 TOWARDS CONVERSION OF QUATERNARY AMINES

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Student

Merve Keser 0862924 BML8R Biochemistry Biology and Medical Laboratory Research Institution RWTH Aachen University Institute for Biotechnology Worringerweg 3 52074 Aachen, Germany

Internship Supervisors

Prof. Dr. Ulrich Schwaneberg Dr. Alan Mertens MSc. Yu Ji Home University Biology and Medical Research Engineering and Applied Sciences Rotterdam University G.J. de Jongweg 4-6 3015 GG Rotterdam, Netherlands Internship Coach MSc. Maarten v/d Velden Internship Period May 2016 to December 2016 Date November 23, 2016


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ABSTRACT Cytochrome P450 monooxygenases are heme containing, diverse, and versatile enzymes that are found in all biological kingdoms. Their function in oxygenation of a substrate has proved them essential in the metabolism of pharmaceuticals and biosynthesis of steroids. Due to their diversity in substrate scope, chemical reactions, and unique physical properties they have been a point in fundamental research in all research disciplines since their discovery in 1958 (1). The general mechanism and function of a cytochrome P450 is the binding of a ligand followed by the binding of di-oxygen and the acceptance of another electron. This forms a reactive complex that is than able to carry out many chemical reactions such as hydroxylation in which the reductive cleavage of oxygen yields an oxygenated organic product and a water molecule (2, 3).

In the interest of their diversity in physical properties as well as chemistry, a cytochrome P450 derived from the Bacillus megaterium (P450 BM3) was impressive in its fused reductase domain property. P450 BM3 possesses a flavin adenine dinucletotide and flavin mononucleotide (FAD-FMN) containing reductase domain that is very similar to eukaryotic P450 reductase and retains a very high catalytic activity in the hydroxylation of fatty acids (4). From this finding, the point of focus became in identification of the amino acids in the active site of P450 BM3 to further enhance this enzyme as well as to gain insight into its function and structure (1).

Harvesting the fundamental functions of enzymes such as their stereo- and regioselectivity, rational and directed evolution strategies are applied to meet the application demands regarding biocatalysts. The OmniChange focused mutagenesis approach applies simultaneous saturation of up to five codons leading to a genetic modification in the amino acid sequence regarding the targeted sites that yield copious amounts of variants (5). The site directed mutagenesis performed by Oliver et al (1) yielded a promising mutant in whose arginine at position 47 within the active site was replaced by glutamate (R47E). This variant of P450 BM3 gave promising results in the catalysis of the unusual P450 BM3 substrate, the trimethylammonium compounds, ranging from carbon chain 12 to 16. However, as noted by Oliver et al (1), this variant showed no loss of catalytic activity to its native substrate scope of fatty acids thus yielding a catalytically superior variant with a broadened substrate scope.

By using the Omnichange method in this research for the saturation of the four codons of the following amino acids L188, Y51, R47, and F87 within the active site of wild type P450 BM3, the hypothesis of generating new mutants that surpass the activity of the already generated R47E mutant by Oliver et al (1) was proposed with the utilization of hexadexyl trimethylammonium bromide (C16 TMA Br) as the main substrate. In performing this research hypothesis, Dr. Alan Mertens and Yu Ji generated approximately 2,000 mutants using the Omnichange method with unknown amino acid substitutions for each mutant at the specific codon saturated sites. High throughput screenings of NADPH consumption- and hydrogen peroxide production activity were applied on the generated 2,000 mutants yielding a total of 7 mutants with improved activity in comparison to that of R47E which was the main control group in this research along with the wild type P450 BM3. GATC Biotech (Cologne, Germany), an external company, conducted the Sanger sequencing of the gene sites that were codon saturated with the Omnichange method, which allowed for the establishment of the amino acid substitutions. Further characterisations of the 7 mutants that were conducted are the determination of total soluble protein content and P450 BM3 protein content assays. Biocatalysis reactions to confirm the efficiency of the 7 mutants towards product formation are a future prospect and are not included in this report, however the method for detection of the product was established and applied with LC-MS using R47E (control) thus the method for product detection was secured but due to time limitations, it was not optimized. This concludes and strengthens the proposed hypothesis of generating a superior mutant to that of R47E that can be utilized fundamentally in the industry by means of sustainability in organic synthesis, metabolism of drugs and catalysis of environmental pollutants that contain trimethylammonium compounds, which can be readily catalyzed without toxic byproducts as opposed to using chemical catalysts (6, 7).


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TABLE OF CONTENTS ABSTRACT ................................................................................................................................................................................ 2

ABBREVIATIONS ..................................................................................................................................................................... 5

1. BACKGROUND: Cytochrome P450 Superfamily ....................................................................................................... 6

2. INTRODUCTION ............................................................................................................................................................... 7

2.1. Catalytic Cycle of P450s ............................................................................................................................................. 7

2.1.1. Cytochrome P450 BM3 ....................................................................................................................................... 8

2.2. OmniChange: Focused mutagenesis ......................................................................................................................... 9

2.2.1. Focused Mutagenesis of P450 BM3 ................................................................................................................ 12

2.2.2. Mutant P450 BM3 R47E .................................................................................................................................... 12

3. PROBLEM AND GOAL OF THE RESEARCH ............................................................................................................ 13

3.1. Problem ......................................................................................................................................................................... 13

3.2. Goal ............................................................................................................................................................................... 14

4. MATERIALS AND METHODS ....................................................................................................................................... 14

4.1. Research Strategy ....................................................................................................................................................... 14

4.2. High Throughput NADPH Consumption Screenings ........................................................................................ 14

Screening of Mutant Library ........................................................................................................................................ 15

4.3. Hydrogen Peroxide Assay ........................................................................................................................................ 16

4.4. BCA Assay .................................................................................................................................................................... 16

4.5. Carbon Monoxide Difference Spectrum Assay ................................................................................................... 17

4.6. Gene Sequencing ........................................................................................................................................................ 18

4.7. Product Detection by Means of LC-MS ................................................................................................................ 19

5. RESULTS ............................................................................................................................................................................... 19

5.1. NADPH Consumtion Screenings ............................................................................................................................ 19

5.2. Hydrogen Peroxide Assay ........................................................................................................................................ 20

5.3. Product Detection by LC-MS .................................................................................................................................. 21

5.4. BCA Assay .................................................................................................................................................................... 24

5.5. CO-difference Spectrum Assay ............................................................................................................................... 25

5.6. Gene Sequencing ........................................................................................................................................................ 27

6. DISCUSSION AND CONCLUSION ............................................................................................................................ 28

7. ACKNOWLEDGEMENTS ............................................................................................................................................... 30

8. REFERENCES ...................................................................................................................................................................... 31

9. LIST OF FIGURES .............................................................................................................................................................. 33

APPENDIX ............................................................................................................................................................................... 35

APPENDIX A: OmniChange Protocol .......................................................................................................................... 35

APPENDIX B: Culturing and High Throughput Screening ....................................................................................... 35


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B.1. Protocol: Preculture and Culturing of Mutants for Screening .................................................................... 35

B.2. Protocol: NADPH Consumption Assay ........................................................................................................... 37

B.3. Protocol: Hydrogen Peroxide Assay ................................................................................................................. 38

APPENDIX C: BCA Analysis Protocol .......................................................................................................................... 40

APPENDIX D: Carbon monoxide Difference assay Protocol ................................................................................. 41

APPENDIX E: LC-MS Method ....................................................................................................................................... 42

APPENDIX F: Raw Data ................................................................................................................................................... 43

F.1. NADPH Consumption Assay .............................................................................................................................. 43

F.2 Hydrogen Peroxide Assay ..................................................................................................................................... 43


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ABBREVIATIONS ACN Acetonitrile

BCA Bicinchoninic acid C12 TMA BR Carbon 12 trimethylammonium bromide C16 TMA BR Carbon 16 trymethylammonium bromide CFE Cell free extract CPR NADPH dependent Cytochrome P450 reductase

CYP Cytochrome P450 DMSO Dimethyl sulfoxide DNA Dioxyribonucleic acid dsDNA Double stranded dioxyribonucleic acid E. coli Escherichia coli EV Empty vector F87 Phenylalanine at position 87 FAD Flavin adenine dinucleotide FMN Flavin mononucleotide GAN G: Guanine; A:Adenine; N: Any nucleotide GDH Glucose dehydrogenase GNN G: Guanine; N: Any nucleotide

HRP Horseradish peroxidase K-‐Pi Potassium phosphate L188 Leucine at position 188 LC-‐MS Liquid chromatography mass spectrometry MTP Microtiter plate

NADPH Nicotinamide adenine dinucleotide phosphate NNK N: Any nucleotide; K: Guanine or Thymine (keto)

nts Nucleotides NYN N: Any nucleotide; Y: Thymine or Cytosine (Pyrimidine)

P450 BM3 Cytochrome pigment P450 from Bacillus megaterium 3 PCR Polymerase chain reaction PTO Phosphorothiolate

R47 Arginine at position 47 R47E Arginine (R) 47 replaced with Glutamate (E) WT Wildtype

Y51 Tyrosine at position 51


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1. BACKGROUND: CYTOCHROME P450 SUPERFAMILY The first P450s were discovered in 1958 in mammalian microsomal liver cells due to their unusual pigment. Their ability to form a complex with carbon monoxide (CO) defined them as a distinct type of heme-binding electron transferring proteins also known as a cytochrome (CYP). The absorptivity shift from the steady state of 420 nm to 450 nm when CO-heme complex is formed gave this distinct cytochrome its name of Pigment 450 (P450) (8).

Cytochrome P450s are part of a superfamily called mono-oxygenases. Their catalytic function is the reductive cleavage of oxygen to form an oxygenated product with water (1). The general reaction catalyzed by P450s is as follows:

RH + O2 + NAD(P)H + H+ è ROH + NAD(P)+ + H2O

The RH represents a typical substrate and the resulting product is the reduced NAD(P)+ and an organic oxygenated (ROH) compound (6). This catalytic system is essential in production of steroid hormones and prostaglandins in mammals and plants (9, 10). It has to be said that the ability of P450s to breakdown drugs, and xenobiotics have paved the way for innovative research. The P450 superfamily is vast although following the course of evolution the general topology and the amino acid sequence alignment between the different P450s is less than 15% (2). This makes it evident that although conserving the overall fold and heme-binding site, there is much diversity in the substrate selectivity. Initially P450s were classified in Class I and Class II enzymes representing prokaryotic and eukaryotic, respectively. However, new research showed deviations of characteristic properties that resembled each other (2, 8). All P450s require the transfer of electrons to catalyze the reaction of oxygenated product and water in which their preference in redox partners plays a big role. Prokaryotic P450 enzymes have a reducing partner that is an iron containing ferredoxin, which is in turn reduced by a flavin adenine dinucleotide (FAD) containing reductase (FDR). Eukaryotic P450s receive electrons from membranous flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) containing reductase, which is also referred to as NADPH-cytochrome P450 reductase (CPR). Mitochondrial P450s utilize adrenal ferredoxin or adrenodoxin with an adrenodoxin reductase. The main objective of microsomal P450s is converting hydrophobic compounds to hydrophilic so that they are easily eliminated or made precursory for further enzymatic cascades (1, 9, 4).

The average molecular weight of cytochrome P450 is 40-60 kDa. The mammalian P450 is synthesized on the endoplasmic reticulum where a single recognition particle inserts the protein into the membrane by an N-terminal peptide anchor during translation (1, 2).

The nomenclature of P450s (CYP) is extensive and is paired to sequence similarities and substrate preference. In order to group a cytochrome P450 gene family together the sequence homology must be >40% and to group genes under a subfamily, the sequence homology must be >55%. CYP is given to designate that it belongs to the P450 gene family (11). Numerals are designated to each P450 gene family starting with 1. Gaps between the numerals are left for the annexation of newly discovered gene families. For example, the P450s involved in steroid biosynthesis fall within families 17 (steroid 17α hydroxylase), 19 (P450 aromatase) and 21 (steroid 21α hydroxylase). The numerals of 11, 51/52 and 100/102 designate the mitochondrial, yeast, and bacterial P450s respectively. Capital letters are assigned for the gene’s subfamily followed by another numeral for the specific gene within that subfamily. For example, after the novel discovery of P450 BM3 in the early seventies by Fulco’s group at UCLA, it was defined by the nomenclature CYP102A1 representing that it is a cytochrome P450 belonging to a bacterial gene family under the subfamily A, with the specific gene 1 (3). Up to current date, according to Nelson et al (12), there has been 35,166 P450 genes identified of which 32,930 genes derived from eukaryotic e.g. animals, plants, fungi, and protozoa, 2,156 genes from bacteria, 52 from archea, and 28 genes derived from virus species (9, 11).


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2. INTRODUCTION 2.1. CATALYTIC CYCLE OF P450S The catalytic cycle of P450s (Figure 1) has been of great interest due to its ability to introduce oxygen into non-active carbon-hydrogen bonds by highly stereo- and regioselective reactions. Cytochrome P450s contain a heme porphyrin that is essential in the chemical cascade making it a part of the active site of the enzyme with surrounding key amino acid residues for the stability of the substrate-enzyme complex (4, 2).

In the resting state of the enzyme, the iron (Fe) atom is at Fe3+ low spin state also known as a ferric state. In this state four nitrogen atoms coordinate the porphyrin ring with the sulfur atom of cysteine bonded to a water molecule in the distal axial position as can be seen in figure 1 indicated by the bold number 1. Upon substrate binding, the resting state of the low spin ferric enzyme is disrupted and the water molecule coordinated at the sixth axial ligand changes the low spin state to a high spin substrate bound complex. (Figure 1: bold number 2). Therefore, the high spin ferric state yields an increased reduction potential that enables, with the help of a redox partner, the concomitant reduction to a ferrous state (Figure 1: bold number 3). Next, the binding of molecular dioxygen produces the ferric-dioxo-P450 complex (Figure 1: bold number 4). This ferric-dioxygen species is readily protonated to produce peroxy-ferric intermediate (Figure 1: bold number 5a). Following the second protonation of the peroxy-ferric intermediate, the reaction yields hydroperoxy-ferric intermediate termed compound 0 (Figure 1: bold number 5b). Upon delivery of a hydrogen atom in the present solvent, the O-OH bond is heterolytically cleaved yielding water and the oxo-Fe4+-porphyrin named compound 1 (Figure 1: bold number 6). Upon reaching towards completion of the catalytic cascade, compound 1 acquires a proton from the substrate to form a ferryl-hydroxy intermediate producing the resting state heme porphyrin and hydroxylated product (Figure 1-bold number 7) (4, 2, 11) .

Fig 1: The general catalytic cycle of cytochrome P450 showing the redox reactions of the heme porphyrin in relation to its redox partner and substrate. Within the cascade, three alternative pathways called shunt pathways are shown to prove possible uncoupling scenarios in regard to enzyme-substrate complex (13).

There are also alternative pathways that occur under physiological conditions. These branching points (see Figure 1: bold numbers 4, 5b, and 6) in the cycle are product inhibiting and are called autoxidation, peroxide- and oxidase shunt pathways in which they render the oxo- or hydroxo- ferrous enzyme back to its resting state without the formation of


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any product. Nonetheless, the enzyme still utilizes its reducing domains and NAD(P)H as its cofactor. Autoxidation results from the cleavage of Fe3+-O bond where the superoxide anion is formed and the enzyme is returned to its resting state. This superoxide anion is reactive and damaging to the enzyme. Therefore, when put in an in vitro reaction, superoxide dismutase must be utilized to neutralize. The peroxide shunt pathway results in the dissociation of the hydroperoxide anion and forms hydrogen peroxide with the addition of the obtained proton from the surrounding solvent. This renders the enzyme back to its resting state and inhibits the transition to the next step of the catalytic cycle for substrate turnover. In order to neutralize the hydrogen peroxide in an in vitro reaction, catalase can be used effectively only when the hydrogen peroxide leaves the active site upon formation, otherwise the accumulation will disfigure the enzyme. In the oxidase shunt pathway the ferryl-oxo intermediate is oxidized to yield water instead of the oxygenation of the substrate and renders the enzyme to resting state without the formation of a hydroxylated product. This shunt pathway phenomenon is known as uncoupling and poses a problem in many of the conducted research (8, 2).

2.1.1. CYTOCHROME P450 BM3 P450 BM3 (see figure 2) was the third cytochrome P450 which was discovered in the gram-positive bacterium Bacillus megaterium and since its discovery attracted a lot of attention due to its redox self-sufficient properties, which is viewed as a novelty in the P450 superfamily (3). It is a soluble fusion protein in which the N-terminal heme domain is linked to a C-terminal reductase domain, showing similarity to that of a eukaryotic P450 CPR containing FMN and FAD domains (1). P450 BM3 is represented by the numerical nomenclature CYP102A1 as previously stated. This enzyme is comprised of a single 119-kDa polypeptide chain that has strong resemblance to eukaryotic P450 moeities thus sparking interest for its use as a model for the mammalian P450 enzymes (3, 14).

The significance of this self-sufficient enzyme applies for example, in the synthesis of organic compounds, degradation of toxic chemicals in the environment, and incorporating plants with resistance to certain pesticides (2, 5). P450 BM3 is located in the cytosol of the bacteria and carries out hydroxylation at the ω-1, ω-2, and ω-3 positions of long chain saturated and mono-unsaturated fatty acids, fatty amides and alcohols, and epoxidation of medium chain mono-unsaturated fatty acids. The preferred substrates that yield a high specific activity are long carbon-chained fatty acids ranging from C14 to C16; it has also given the highest turnover rate from that of any other P450 monooxygenase. Furthermore, because of the strong resemblance to that of eukaryotic microsomal P450s namely the fatty acid hydroxylase (CYP4A) family, it enables more insight into the catalytic and structural properties of this categorized enzyme in both kingdoms. Due to its self-sufficiency and broad range of substrates, it is an ongoing topic of fundamental research to broaden the perspective of its applicability (1, 4, 10).

Fig 2: Tertiary structure of the P450 BM3 protein. The heme domain is shown here in blue in which the heme plane in the active site is shown in red. The FAD domain is presented in green and the cofactor FMN is presented by yellow. 458 and 479 are the last visible residues in the


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heme domain and FAD domain, respectively (16). This image represents the monomer and thus the single polypeptide chain that make up the reductase and the heme domain however, the quaternary structure of this protein is comprised of two monomers thus having two substrate entry sites. The positioning of the heme plane in this figure is of importance considering relation of the entry site of the substrate in proximity to the heme.

2.2. OMNICHANGE: FOCUSED MUTAGENESIS The functions of enzymes are not only precise in stereo and regio-selectivity but it is a naturally occurring phenomenon that an enzyme does not need an external compound to generate this level of precision in product formation. This precision of enzymes is in comparison to chemical reactions that require an additional chemical catalyst to yield the wanted stereochemistry of the desired product. Enzymes are efficiently functional in an optimal temperature, within a defined pH range, at a certain salt concentration and in an aqueous solution. Enzymes are proven to be a natural resource of catalysts and this gives way to creative advanced ideas in improving the catalytic activity that can fundamentally be applied to all processes because generally, the natural maximum potential of enzymes are hindered when placed industrial applications that require large amounts of yield with minimal run-down. In order to advance the catalytic potential of enzymes of interest, rational design and directed evolution are the groundbreaking principles. The OmniChange method (See Appendix A for protocol) provides a practical and robust protocol to simultaneously saturate up to five (albeit less than 5 is also feasible) codons without the necessity of DNA restriction or ligation enzymes. This method is fully independent of sequence or fragment size, and yields high genetic diversity in one PCR step (5). The quality of this method comes from the design of primers with 12 phosphorothiolated (PTO) nucleotides in which the sulfur atom replaces one of the oxygens in the phosphate linkage between bases in the oligonucleotide (See Figure 3). This allows for the quality of an efficient cleavage of the phosphorothiolated nucleotides using iodine and ethanol thus omitting the use of any additional DNA restriction enzymes. Therefore, cutting time and increasing efficiency in generating inserts for the hybridization with the vector (17).

Fig 3: In section A, the upper molecule shows the natural nucleotide backbone of DNA, which is connected via phosphodiester bonds. The lower molecule is the phosphorothiolated DNA backbone in which the sulfur atom is indicated by an asterisk. In section B, the chemical cleavage of phosphorothiolated nucleotides with iodine and ethanol is shown under pH 9.0 (17).

For clarification of the strategy of the state of the art Omnichange method, the main element is the selection of the target codon for mutation and the hybridization area upstream or downstream of the targeted codon as seen in Figure 4, 3A and 3B (17). Followed by the amplification of fragments and vector by standard PCR. The PCR highlights the paramount significance of this method. The primer design for the PCR consists of phosphorothiolated nucleotides containing the NNK codon for saturation at the selected target site (see Table 1 for nucleotide nomenclature). NNK signifies that the first two positions, N and N, of the targeted saturation site codon can be of any nucleotide, and the third position, K, of the codon must be a Guanine or a Thymine An example of a forward and a reverse primer design scheme is 5’-12 (nts)*-NNK-15 (nts)- 3’ amounting to a total of a primer with 30 nucleotides in which the first 12 nts,


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given with an asterisk in the scheme, at the 5’ start have phosphorothiolated backbones, and the NNK codon followed by the rest of the 15 nucleotides are the usual phosphodiester bonds found in DNA and RNA (see Figure 4, 3C). NNK codon is generally used for the maximum amount of genetic diversity possible by generating only one stop codon, the UAG, out of the total three stop codons which are the UAG, UAA, and UGA to prevent the termination of translation once transformed into the host cell. The 5’-12 PTO nts are complementary to the selected hybridization area thus ensuring the hybridization of the fragments and the vector after the cleavage step. PCR yields the amplification of the fragments bearing the NNK saturation site. Instead of the use of restriction enzymes, iodine ethanol solution is used to cleave each 12 consecutive PTO nts, which results in the removal of all of these 12 nts. This creates 5’ DNA overhangs in each fragment with specific 12 nt long hybridization sites. After the hybridization of the vector and fragments, a full-length plasmid with nicks in the DNA backbone at each hybridization site is formed bearing up to 5 NNK codon saturations. These plasmids are then transformed in to E.coli where its nick-repair system is exploited for repair and plasmid replication (5, 17).

This process is shown in application in figure 5 bold number 1 where five target codons and hybridization areas are selected in a vector. A1, B, C, D, E and A2 designate these areas with their respective primers bearing the 5’-12 PTO nts and the NNK codon for saturation. Once the amplification by standard PCR is conducted (Figure 5 bold number 2), the fragments or inserts are cleaved with iodine solution resulting in 5’ 12 nts long DNA overhangs (Figure 5 bold number 3). The overhangs ensure correct hybridization and the formation of a plasmid with all of the inserts and their respective NNK saturated sites. After hybridization, transformation into a host cell is necessary to exploit the nick-repair system and replication of the plasmid (5).

Fig 4: Scheme of efficient oligonucleotide design using OmniChange method where the selection of the codon is targeted (3A). 12 nts are marked upstream the targeted codon for the designation of hybridization and complementary 12 PTO primer design (3B) The design of oligonucleotides with each of the 12 consecutive phosphorothiolated nts are introduced on the 5’ ends of each primer followed by the NNK codon and the rest 20 nts which are bonded by the usual phosphodiester bonds found in DNA and RNA yielding a total of 35 nts long forward primer in (3C) (17).


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TABLE 1: IUPAC NUCLEOTIDE NOMENCLATURE FOR CODON SATURATIONS

Fig 5: The Omnichange method scheme. 1) The amplification with PTO primers by means of standard PCR is done to simultaneously saturate up to 5 codons by using the NNK codon to generate genetic diversity in the selected positions. Followed by cleavage of the 12 PTO nts to create 5’ DNA overhangs in which correct hybridization is secured. Hybridization generates full-length plasmids with inserts ready for transformation into a vector (17).

The application of such a method generates a vast amount of mutations in selectivity and activity therefore yielding countless mutants (e.g. ≤5 target sites x NNK= 205 =3.2 million protein variants) that may or may not have improved qualities. The saturation of codons and thus insertions of mutations are done by the design of PTO primers using nucleotide nomenclature (see table 1). Ultimately, high throughput screenings must be carried out to determine the


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activity of the improved variants with reaction conditions befitting the enzyme of interest and gene sequencing must be done to determine the amino acid substitutions resulting from the nucleotide codon saturation e.g. NNK.

2.2.1. FOCUSED MUTAGENESIS OF P450 BM3 Enhancement of the activity and selectivity of an enzyme is dependent on the interaction with its substrate. More importantly, the residues within the substrate-binding pocket give insight on the stability and efficiency of the substrate-enzyme complex. Considering the peculiar structure of the P450 BM3, acknowledged for having a fused reductase domain that is similar to its eukaryotic counterparts, the self-sufficiency of this enzyme is an interesting factor for its refinement in activity and/or selectivity making it attractive for site directed mutagenesis. While its most preferred substrates are that of saturated long chain fatty acids, through mutagenesis, the substrate scope can be broadened. The most important residues in substrate recognition within the substrate pocket are the arginine 47 (R47) and phenylalanine 87 (F87) in which the arginine 47-sidechain is positioned on the entrance of the substrate pocket to act as a stabilizer upon entry and binding (Figure 6). The Van der Waals interactions of the aromatic ring of phenylalanine 87 with the heme are of importance in the formation of the hydrophobic pocket for seclusion of the terminal methyl end of the fatty acid to protect it from oxidation thus proving also another importance in the regioselectivity of the cascade (1, 5). Along with these two amino acid residues, tyrosine 51 (Y51) and leucine 188 (L188) also prove to be significant in the stabilizing interaction with the substrate and the selectivity of substrate chain length, respectively (10, 14, 15). The focused site mutagenesis of these four key amino acid residues in the active site of P450 BM3 proposes the potential to enhance the activity and performance of this enzyme towards a given substrate. In the generated library of Dr. Alan Mertens and Yu Ji, they implemented the following saturation codons in their primer design: NNK, NYN, GAN, and GNN for these key amino acid positions L188, Y51, R47, and F87 respectively.

Fig 6: Active site structure within the substrate binding pocket and surrounding significant amino acid residues. This structure was defined with the use of palmitoleic acid (shown in ‘color’) as substrate. As present, R47 and Y51 are positioned in the opening of the substrate access channel regulating the interaction with the substrate for stabilization upon binding and substrate induced heme iron spin shift. The position of F87 is in accord to its responsibility of shielding the terminal methyl end from any hydroxylation or oxidation. L181 being positioned in the middle of the substrate entrance channel is synergistic with its role of regulating the carbon chain length of substrate (1, 7).

2.2.2. MUTANT P450 BM3 R47E Site directed mutagenesis is applied to replace the R47 with glutamate (R47E) in P450 BM3. This mutation results in the exchange of a positively charged arginine (R) at amino acid position 47 to a negatively charged glutamine (E) to broaden the substrate scope of P450 BM3 towards positively charged alkyl trimethylammonium compounds. This mutagenesis first conducted by Oliver et al (6) yielded very promising results in the catalysis of carbon 12 (C12) to carbon 16 (C16) alkyl trimethylammonium compounds yielding hydroxylated product at the ω-3, ω-2, and ω-1 positions (Figure 7). Alkyl trimethylammonium compounds are naturally poor substrates for the wild type P450 BM3 enzyme (see Figure 8 for turnover values). Increased activity resulting from the site mutagenesis of R47E towards this unusual substrate did not hinder the P450 BM3 of its activity towards its naturally preferred fatty acid substrates. R47E has been established


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by characterization through sequencing, mass spectrometry, and kinetics on substrate binding (6). Alkyl trimethylammonium compounds are also known as quaternary ammonium compounds that have a positive charge with the structure of NR4+ in which the R is the rest group being an alkyl or aryl groups that are widely used as surfactants, detergents, and disinfectants (16, 17). The use of these compounds with P450 BM3 is to show a fundamental potential in the application of site directed mutagenesis to broaden its substrate scope and interaction so that its significance can be proven in not only organic chemical synthesis but also beneficial in the identification and elimination of environmental pollution concerning comparable compounds.

Fig 7: Molecule shown is the substrate positively charged C16 trimethylammonium bromide hydroxylated in the positions ω-1, ω-2, and ω-3. P450 BM3 R47E variant at one position per reaction cycle hydroxylates the substrate.

Fig 8: Kinetic constants correlating the wild type P450 BM3 with that of P450 BM3 R47E mutant. In the seventh column with the constant kcat/Km it is clear that the mutant activity on the substrates C12-C16 TMA give a higher turnover frequency when compared with the wild type (6).

3. PROBLEM AND GOAL OF THE RESEARCH 3.1. PROBLEM The research of Oliver et al. (6) in site directed mutagenesis of P450 BM3 as mentioned above, has provided subsequent evidence in which while maintaining its catalytic activity towards saturated fatty acid substrates such as laurate, myristate, and palmitate, the P450 BM3 mutant R47E has also increased catalytic activity towards alkyl trimethylammonium compounds ranging from 12 to 16 carbon long chain size. Although the R47E is a more than satisfactory functioning mutant, in the research of Oliver et al (6), only one position being the R47 is saturated to glutamate (E) to prove that not only does the mutant maintain its native fatty acid substrates but also show an increased activity towards quaternary amines. Oliver et al (6) is also the only unique research conducted regarding quaternary ammonium compounds as substrates with subsequent full characterization of the mutant P450 BM3 R47E. Taking the four key amino acid positions, R47, F87, Y51 and L188, that are of significance in the active site of the P450 BM3 as mentioned before, using the Omnichange method in the saturation of these four key amino acid positions brings forth the hypothesis of the potential to generate an overall better performing mutant. In regard to this, the challenging new step is by using the state of the art method OmniChange to generate new mutants that surpass the catalytic activity of


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R47E. Accompanying this pursuit are the aspects of uncoupling correlated to the shunt pathways present in the P450 catalytic cycle as discussed before that affect product formation. The best-performing variant generated by the Omnichange method will provide insight into which amino acid substitutions, attained from each of the four key amino acid residues, affects the function of its role within the active site and also demonstrates an overall effect on the catalytic activity towards quaternary amine substrates. The discovery of a better performing mutant than that of R47E with further characterizations to define its enhanced properties will fundamentally allow for its use to be made in research and/or industries such as pharmaceutical, organic synthesis, and environmental technology for sustainable biotechnological processes.

3.2. GOAL The foremost goal of the research is to establish a new mutant that has a higher catalytic activity than that of the R47E. In pursuit of discovering the best performing mutant, a library of approximately 2,000 mutants were generated by Yu Ji and Dr. Alan Mertens using the OmniChange method by saturating the four key amino acid residues of R47, Y51, F87, and L188. As stated before, when generating their library they implemented the following saturation codons in their primer design: NNK, NYN, GAN, and GNN for the amino acid positions L188, Y51, R47, and F87 respectively to yield a better performing mutant towards quaternary amines. The goals for the research project given to me are:

1. The high throughput screening of the generated library by means of indirect enzyme activity measurement of the consumption of NADPH.

2. Detection of H2O2 production that results from the peroxide shunt pathway. 3. The determination P450 BM3 protein content. 4. The analysis of plasmid gene sequencing conducted by GATC Biotech (Cologne, Germany) for the determination

of the amino acid substitutions at the four key amino acid saturation sites. 5. The use of LC-MS with the corresponding method developed by Yu Ji, for the detection and confirmation of

product formation by the generated variants.

4. MATERIALS AND METHODS All chemicals were acquired from the Sigma Aldrich and laboratory equipment supplier www.VWR.com.

4.1. RESEARCH STRATEGY The project was divided into four stages. The first stage began with the optimization of the NADPH consumption assay for the determination of the most feasible activity of the positive control R47E mutant. The Second stage was the screening of the mutant library using the optimized NADPH consumption assay to select the most significantly active mutants that match or surpass the activity rate of R47E towards trimethylammonium compounds. Once the best mutants were determined, they were re-screened by performing the peroxidase assay, followed by plasmid gene sequencing and product detection by means of LC-MS for further characterization.

4.2. HIGH THROUGHPUT NADPH CONSUMPTION SCREENINGS In order to determine the NADPH consumption and thus the activity of the enzyme, the assay was first optimized with the mutant P450 BM3 R47E variant enzyme and then applied to the generated library (See Appendix B.2 for protocol). The substrate that has provided significant results was the hexadecyl trimethylammonium bromide (C16 TMA Br). The cell pellets that were acquired from the protein expression were re-suspended with 100 mM potassium phosphate buffer (pH 8), (5 g/L) lysozyme solution, and incubated for an hour in the 37 degrees Celsius microtiter plate shakers at 900 rpm. Followed by centrifugation, the acquired supernatant was the cell free extract (CFE) that was further used in the assay. 25 ml of 1.25 mM C16 TMA Br stock solution was made by weighing 11.4 mg of C16 TMA Br and further dissolved in 25 ml of the organic solvent DMSO. A fresh batch of 0.8 mM NADPH was prepared with 100 mM potassium phosphate buffer (pH 8). Control and sample pipetting schemes were carried out as follows:


15

TABLE 2: PIPET SCHEME OF NADPH CONSUMPTION ASSAY Compounds Sample Control Notes DMSO with Substrate 1.25 mM

10 µL (with substrate) 10 µL (Only DMSO) Final conc. 50 µM (diluted 1:25)

100 mM KPi Buffer 155 µL 155 µL CFE 35 µL 35 µL Mix well! After incubating the substrate with the P450-BM3 (mutant) enzyme for 5 minutes, the conversion of NADPH was started by adding:

NADPH 0.8 mM 50 µL 50µL Final Conc. 160 µM (diluted 1:5)

Total 250 µL 250µL The screening was done using 96 well flat-bottomed MTP and a MTP spectrophotometer (TECAN Sunrise) in which the absorbance was recorded at 340 nm. First the plate was measured end-to-end without the addition of NADPH but only with substrate solution and CFE. Once the NADPH was added the kinetics was set at 13-second intervals with 70 cycles. This was repeated with the control plate. The background (control) data and sample data were manually analyzed and their trends determined.

SCREENING OF MUTANT LIBRARY From the acquired library of approximately 2,000 P450 BM3 mutants that were stored in 20- 96 flat- bottomed MTPs containing LB medium and glycerol were given the name master plates with assigned numbers 1-20. A pre-culture was made first (see Appendix B.1 for pre-culture protocol) followed by an expression culture according to the protocol for hydroxylases (22). Pre-culturing was done by pipetting 150 µl LB medium enriched with kanamycin, for growth selection, in each of the 96 wells of the V-bottom MTP. Ratio of kanamycin and LB medium volume was set to 1:1000. Next, 4 µl of mutants from each well of the master plate were transferred to the pre-culture MTP and incubated overnight in the 37 degrees Celsius MTP shaker at 990 rpm with 70% humidity. Expression culture was done using 96 deep-well plates by pipetting 600 µl of TB medium enriched with IPTG, trace element solution, and ALA; which are the protein expression inducing agents. The ratio of TB medium and the protein expression inducing agent volumes are set to 1:1000. 4-5 µl of the pre-cultured mutants in each 96 wells of the pre-culture plate is transferred to the expression deep-well plate and incubated for ~20 hours in the 30 degrees Celsius MTP shaker at 500 rpm with 70% humidity. Next, the cultured plates were centrifuged under 4 degrees Celsius at 4,000 rpm followed by the removal of the supernatant. In order to gain the CFE, the cell pellets were first washed with 75 μl KPi Buffer (100 mM, pH 8.0) and then treated with 125 μl lysozyme (5 g/L) and incubated for 1 hour at 37 degrees Celsius in the MTP shaker. Followed by the incubation, the MTP plates were centrifuged under 4 degrees Celsius at 4000 rpm. The CFE was the resulting supernatant.

Each well in each plate of a library represents a different mutation in the P450 BM3 enzyme. The screening was the same as stated above, using the C16 TMA Br as the main substrate. Each screen was measured under 340 nm and the trend of the slope was analyzed following the selection of those that perform best. From the preliminary screenings of all 2,000 variants, those that yielded an increase in slope to that of the control group R47E were selected and another round of screening was repeated. From these results, again the selections of best performing variants were made and the screening process was repeated. This confirms many consecutive rounds of variant selection and the repetitive screening of the chosen variants. Following this consecutive screening manner, the selections of variants were narrowed down to the 7 top best. The selections of variants were not signified by a statistical significance test because this assay was an indicative method for the search of the best performing variants compared to that of R47E and the supervisors also did not propose such a statistical test. By the comparison of variant activity in terms of slope to that of R47E, it strongly indicated a better performing variant and the consecutive screening data were also in accordance to each other.


16

4.3. HYDROGEN PEROXIDE ASSAY Hydrogen peroxide (H2O2) formation is a possible product resulting from the peroxide shunt pathway of the catalytic cycle of P450. Peroxide is formed within the active site and is detected when it is released. Hydrogen peroxide is generally carried out of the active site upon formation, however, if it is not carried out from the active site the accumulation of this product leads to the damage of the enzyme, e.g. by denaturation. Even when it is carried out of the active site, it still poses a threat to the overall enzyme because hydrogen peroxide is a highly reactive agent, thus having the potential to react with other parts of the enzyme complex. In order to detect the amount of hydrogen peroxide, horseradish peroxidase (HRP) and Amplex Red™ are key component of this assay. HRP is a peroxidase that utilizes H2O2 as cofactor for the oxidation of many organic and inorganic compounds in which the H2O2 is converted to water: neutralizing the reactivity of H2O2 in coupled enzyme assays (23). In the presence of HRP, Amplex Red ™ is an organic substrate that is catalyzed with a stoichiometry of 1:1 to form a highly fluorescent resorufin. Protocol in Appendix B.3 was followed in carrying out the hydrogen peroxide assay (variations were made in the protocol regarding the volume and equipment for the first part of the assay). Pipet scheme is as follows for the hydrogen peroxide assay:

TABLE 3: VOLUMES FOR NADPH CONSUMPTION Compound Volume (μl)

CFE 10

C16 TMA BR (1.25mM) 5

KPi Buffer (pH 8.0, 100mM) 65

NADPH (1 mM) 20

Total 100 The absorbance was measured at 340 nm on the TECAN Sunrise instead of TECAN Infinite M200/1000 in 30-second intervals lasting up to 15 minutes and finally ending the reaction with 20% v/v acetonitrile. The second part of the reaction was done by adding 125 μl of the Amplex Red™/HRP stock solution (2 U HRP and 8μM Amplex Red ™) and was measured by the TECAN Infinite M200/1000 at a fluorescence of 535/590 nm with intervals of 30 seconds lasting up to 30 minutes. The fluorescence of NADPH consumption was not measured by the TECAN Infinite as stated in protocol due to high deviations within the values given in fluorescence units when measuring the fluorescence of NADPH, however, no extreme deviations were given when hydrogen peroxide was measured. The slope of both trends were determined and graphed together to show the yield in peroxide with contrast to the NADPH consumed in the reaction thus providing insight into the coupling efficiency.

4.4. BCA ASSAY In order to determine the total protein content of an expressed culture or library, a protein assay must be applied. The bicinchoninic acid (BCA) based assay was done by utilization of biuret reaction, which is the reduction of Cu2+ to Cu1+

in an alkaline medium. The bicinchoninic acid-containing reagent determines the ultra sensitive and selective colorimetric detection of the cuprous cation. The binding of the BCA with one cuprous ion yields a purple color that is detectable at an absorbance of 562 nm. This color detection gives a linear trend on par with the increasing protein content ranging from 20-2000 μg/ml (24).

To perform the BCA assay, flask expression of the 7 chosen variants, R47E, and WT was made in order to attain the CFE of all of the samples. First a pre-culture was made by enriching 4ml of LB medium with 4 μl Kanamycin in reaction tubes followed by transferring 4 μl of the selected variants from the respective MTP master plates that were stored in -80 degrees Celsius. The culture was grown overnight at 900 rpm and 37 degrees Celsius. From the pre-culture in reaction tubes, flask expression was made for each sample by inoculating 50 ml of TB medium with 50 µl Kanamycin and 500 µl of the overnight culture in 100 ml erlenmeyer flasks. The flasks were incubated at 30 degrees Celsius, 250 rpm untill the optical density (OD) at 600 nm reached between 0.8 and 1.0. The OD at 600 nm had reached 1.3 after 3 hours and the cultures were supplemented with 50 µl ALA, 50 µl Thiamine, and 50 µl IPTG for inducing expression. After the addition of the supplements, the flasks were incubated for 24 hours under 30 degrees Celsius at 250 rpm (See Appendix B.1 for Flask Expression protocol). Cultures in the flasks were transferred to 50 ml falcon tubes and centrifuged at 4000 rpm under 4 degrees Celsius followed by the removal of the supernatant. Cell lysis and crude


17

extract preperation was done by first washing the cells with 10 ml KPi Buffer (100 mM, pH 8.0) followed by the addition of 3 ml lysozyme (5g/L). Next, the falcon tubes conatining the cultivated cultures were incubated at 37 degrees Celsius at 250 rpm for 30 minutes. After incubation, the falcon tubes containing the culture were sonicated and centrifuged again for 30 minutes at 4 degrees Celsius and 4000 rpm. The acquired supernatant resulted in the CFE (See Appendix B.1 for full CFE preperation protocol). This overall protein content determination assay was conducted according to the kit’s specifications and protocol; deviations that were made to the provided protocol are stated in Appendix C. The BCA assay is necessary for providing insight on the level of protein expression by determining the overall protein concentration in the expressed culture. In figure 9, an example of the color precipitation of the assay done on the expressed variants (samples) and the linear color gradient of the standards are presented.

Fig 9: An example of the BCA Colorimetric assay on MTP with standards ranging from 20 to 2000 µg/ml on first three rows. At the bottom right hand is the samples in three dilutions ranging from 1:5, 1:8, and 1:10 done in triplicates. MTP is scanned by TECAN Sunrise spectrophotometer at 562 nm.

The BCA assay was applied to all 7 best performing variants, R47E and WT (samples). The samples were diluted in ratios of 1:5, 1:8, and 1:10. Each of the standards and samples were assayed in triplicates. Absorbance at 562 nm given by the standards were averaged and graphed to yield a standard curve with the respective equation. The standard deviation and the average of the absorbance values given by the dilution factors, 1:5, 1:8, and 1:10 of the samples and control units were calculated and graphed for an overview in the expression of total soluble protein within the expressed culture.

4.5. CARBON MONOXIDE DIFFERENCE SPECTRUM ASSAY Cytochrome P450 is a superfamily of enzymes that have the capacity to catalyze the monooxygenation of a variety of natural and unnatural substrates. The noncovalent bound porphyrin ring allows for these enzymes to have a characteristic spectrum. Upon the reduction of this heme group, cytochrome P450s are able to bind carbon monoxide on its sixth ligand. Binding of carbon monoxide (CO) to the heme group of cytochrome P450 effectively determines the presence of the correctly or incorrectly folded protein. The carbon monoxide binding assay is also useful in determining the cytochrome P450 concentration in an expressed culture or library. The non-reduced form of cytochrome P450 gives a peak at 420 nm. Only when it is reduced it can be bound to CO thus forming a peak at 450 nm. The binding of CO confirms the correctly folded protein that allows for the ligand CO to reach its active site. If the folding of the protein is incorrect, the peak at 450 nm will be absent indicating that the heme group is not reduced and therefore is unable to accept a substrate (25).

The P450 protein content was first determined with the R47E expressed culture as a control unit by pipetting 1 ml of the CFE into a plastic cuvet and the measured spectrum was recorded as the baseline. Next, sodium dithionite was added as a reducing agent and the measured absorbance spectrum was recorded as “reduced”. Followed by the gassing of the cuvet containing the reduced CFE with carbon monoxide for 30 seconds under a fume hood, the absorbance


18

spectrum was measured again with the respective recording of “reduced and gassed”. This step was repeated consecutively until the measured absorbance spectrum peak at 420 nm dissipated and a maximal peak at 450 nm formed indicating the bound CO. This assay is further applied to the 7 top best performing mutants after the large-scale (flask) expression as stated in 4.4 (also See Appendix B.1 for protocol Flask Expression). After the maximal peak at 450 nm was detected, gassing is discontinued and the concentration of P450 was calculated by the alteration of Lambert-Beer equation and the molecular weight of P450 BM3 (See Appendix D for full protocol):

Law of Lambert-Beer: 𝐴 = 𝜀 ∗ 𝑙 ∗ 𝐶

A: Absorbance in nm Ɛ: Molar extinction coefficient in M-1 cm-1 l: path length of light C: Concentration of the analyte MW P450 BM3: 119,000 Da

4.6. GENE SEQUENCING Plasmid extraction was done from the CFE’s of the 7 chosen variants in accordance to the protocol provided by the manufacturer of the kit Nucleospin® DNA, RNA and Protein Purification (Macherey Nagel, Düren, Germany). The isolated plasmids were sent to GATC Biotec (Cologen, Germany) for sequencing. GATC Biotech (Cologne, Germany) specializes in many genomic techniques such as sequencing, liquid biopsy, epigenetics, and transcriptomics among which the Sanger sequencing was used for the sequencing of the variants. Sanger sequencing is also known as chain-termination sequencing in which di-deoxynucleosidetriphosphates (ddNTPs) are used to terminate the DNA elongation in in-vitro DNA replication. These ddNTPs lack the 3’-OH group, as opposed to the deoxynucleoside triphosphates (dNTPs), that are required for the formation of a phosphodiester bond in DNA backbone thus when ddNTPs are incorporated, DNA polymerase is unable to elongate the strand. In the first step of this sequencing reaction, the DNA to be sequenced was denatured by application of heat in to two single strands: the template strand and the complementary strand. Next, the T7 promoter primer (Thermo Fischer Scientific, Catalog #N56002) derived from the T7 bacteriophage was provided along with the sent samples and consequently utilized during the Sanger sequencing by means of binding to the template DNA strand. T7 promoter primer is complementary to the template strand and has the sequence “5’ TAA-TAC-GAC-TCA-CTA-TAG-GG 3’ ” in which the third G upstream from the 3’-end on the primer sequence corresponds to the first base transcribed by the T7 DNA polymerase from the bacteriophage T7. T7 promoter primer is strongly used in sequencing due to its strong affinity and efficiency with T7 DNA polymerase (26, 27). The T7 DNA polymerase is of importance because in comparison to other polymerases its discrimination to dideoxynucleotides is minor and thus a lower concentration of ddNTPs is feasible in addition to its high processivity, T7 DNA polymerase has a strong 3’-5’ exonuclease activity in which when proofreading, it excises incorrectly base paired nucleotides to the DNA template (27). This is an unwanted characteristic when sequencing. In current Sanger sequence techniques, genetically altered T7 DNA polymerases are used in which the exonuclease activity is inactivated (26, 28).

Once the complementary T7 promoter primer was bound to each template strand in each reaction, the DNA synthesis reaction was initiated from the 3’ end. Followed by the addition and consecutive incorporation of the dNTPs in a template dependent manner. In each reaction four fluorescently dyed ddNTPs that represent each DNA base were added and incorporated by the T7 DNA polymerase. The incorporation of ddNTPs terminates the elongation of DNA as mentioned before and as a result, many copies of different length DNA fragments were generated in each reaction: terminated at all of the nucleotide positions by one of the ddNTPs. The reaction mixtures were automatically loaded onto capillary electrophoresis tubes in which the DNA molecules were separated by fragment size. The DNA sequence was determined by the detected fluorescent emission as it flowed through the gel.

The sequence data was analyzed with the program “BioEdit Alignment Editor” in which all the DNA sequence(s) of the selected mutants were aligned and compared to that of the P450 BM3 wild type genome. The first 60 base pairs (bp) in the DNA sequence read are unreliable therefore the annealing of the T7 primer was placed 60 bp upstream of the sequence of interest. This results in a 60 bp shift of the mutated amino acid codon base position in the sequenced DNA, for example; R47, (46X3)+60= 201st nucleotide base pair designates the amino acid arginine in the sequence read instead of nucleotide base pair position of 141. By comparing the sequenced genomes of the variants at the saturated site base pair position to that of P450 BM3 wild type, the identification of the amino acid substitutions were made.


19

4.7. PRODUCT DETECTION BY MEANS OF LC-MS The screening methods, and identification of protein allows for the confirmation that not only the expression is successful but also the rate of reaction is at a speed that may or may not surpass the controls. However, in terms of product production, these assays do not yield quantitative results. To quantify and unify all the evidence leading to a successful mutant, a product detection method is required. In collaboration with Dr. Smith from the Department of Biology at RWTH Aachen University, the development of the LC-MS method was first done with the control group R47E so that the method could be established and optimized for further applications on the variants. However, due to time limitations, only the method was developed and optimization of this method was not finalized. By this developed method, the peaks of substrate and product are roughly determined and unfortunately, no computer image was available from Dr. Smith. (See Appendix E for full protocol).

R47E was first collected in a 1.5 ml eppendorf tube from the reactions carried out by the NADPH consumption assay using the CFE of R47E. The extraction of substrate and product from the reaction was done by the addition of chloroform and potassium iodide (KI) salt to yield a two-phase system. This was further sonicated for 3 minutes with an amplitude of 40%, followed by centrifugation at room temperature at the highest speed. The resulting sample yielded a two-phase system in which the organic phase contained the extracted substrate and the product; the aqueous phase contained the rest of the water-soluble proteins found in the CFE along with NADPH. The organic phase was transferred to a new 1.5 ml eppendorf tube and the addition of chloroform with the subsequent sonication step was repeated with the remaining aqueous phase so that a successful extraction of all the substrate and product from the reaction was guaranteed. Both organic phases from the first and second round of the addition step of chloroform for extraction were combined. 100 μl of the total organic phase sample was transferred to glass GC inlets and placed in a glass GC vial in preparation for the injection to the LC-MS. The LC-MS gradient elution method was applied upon injection with the mobile phase of solvent A with ratio of 20:80 ACN and water with 1% acetic acid, and solvent B with the ratio of 95:5 water with 10 mM ammonium acetate (See Appendix E for LC-MS protocol) (21).

5. RESULTS 5.1. NADPH CONSUMTION SCREENINGS NADPH consumption screenings were done according to the protocol found in Appendix B.2. Approximately 2,000 (20x96-wells of the MTP) mutants have been screened and analyzed. Each well in the MTP plate represents a different mutant from the OmniChange library that has been generated before the start of this project. Screenings were carried out in triplicates and the slope of the NADPH consumption was determined by absorbance at 340 nm over time with its corresponding standard deviation. The results were compared to the control units of R47E, WT, and Blank (EV).

The results shown here are the final screenings of the 7 top best performing mutants that were selected from the consecutively conducted preliminary screenings. The data values are represented by the plate number (Pl) and corresponding well number, ranging from B2 to G11. The slope is determined by the first few cycles to show initial velocity for the rate of NADPH consumption by the P450 BM3 variants at an early time before all of the NADPH is utilized and the trend reaches a plateau. This is to determine the optimal activity that takes place when the entire enzyme is saturated with the substrate to form the ES complex and proceed further into its reaction cascade by utilizing its cofactor, which in this case is NADPH. The NADPH consumption trends of the 7 chosen variants are presented in the Appendix F.1 by figure 10B.


20

Fig 10: Slope of the NADPH consumption of the chosen variants regarding the NADPH Consumption assay. Pl 11 H11 and Pl 19 G5 yield the highest slope in comparison to that of R47E, showing a higher activity than that of the control group (R47E).

In figure 10, clear increases in the slope of the chosen variants are seen in comparison to that of R47E. Those that show an equal or lower value in slope still have potential due to their promising NADPH consumption trends given in the Appendix F.1 in regard to the NADPH consumption trend of R47E.

5.2. HYDROGEN PEROXIDE ASSAY Hydrogen peroxide assay was carried out as stated in Appendix B.3 with the deviations as stated in Chapter 4.3. The selected 7 best performing variants for the final screening of NADPH consumption were also run through the hydrogen peroxide assay. Thus, the comparison of the NADPH consumption and hydrogen peroxide production can be made and the coupling efficiency strongly indicated. Low hydrogen peroxide and high NADPH consumption is linked to better coupling efficiency whereas the opposite suggests uncoupling and possible enzyme denaturation due to the accumulation of hydrogen peroxide. The results presented here are the hydrogen peroxide formation of the top 7 best performing variants. The hydrogen peroxide formation trends of the seven chosen variants are presented in Appendix F.2 by figure 11B.

In figure 11 the comparison of the slopes of hydrogen peroxide production and the NADPH consumption to R47E are shown in percentages. Mutants that show more than a 30% decrease in their slope for hydrogen peroxide formation when compared to the slope of NADPH consumption, indicate a forecast in low uncoupling circumstances towards the yield of the product. The lower the slope percentage of hydrogen peroxide, and the higher the slope percentage of NADPH consumption, the stronger the indication of the coupling of the enzyme to product formation. Pl 2 E7 and Pl 6 A4 do not show high percentages of NADPH consumption and are on par to the yield of R47E, however the high ratio of hydrogen peroxide formation and NADPH consumption indicates efficient coupling to product formation. Pl 10 H11, P11 H11, and P19 G5 clearly show a high ratio between hydrogen peroxide production and NADPH consumption thus indicating a clear efficiency towards product formation without uncoupling and thus a potential in a better performing mutant than that of R47E.

0

0.0005

0.001

0.0015

0.002

0.0025

0.003

ΔA/Δ

t

Slope of Selected Active Mutants NADPH-consumption (First 90 s)

Library Pl 2-19 Pl 2 E7

Pl 6 A4

Pl 8 G6

Pl 10 H11

Pl 11 H11

Pl 12 F3

Pl 19 G5

R47E

WT

EV


21

Fig 11: The hydrogen peroxide formation in comparison to that of NADPH consumption ratios for each variant are given in percentage in regard to R47E as benchmark.

5.3. PRODUCT DETECTION BY LC-MS The molecular weight of non-hydroxylated alkyltrimethylammonium bromide is 364.45 (Figure 7) Da with positive charged nitrogen at the N-terminal. The base molecular weight without the bromide ion is 283.50-284.50 Da. When hydroxylated by the P450 BM3, an OH group will be added to either of the ω-3, ω-2, ω-1 positions on the C-terminal which would increase its molecular weight by ~16 Da. In figure 13 the five rows of chromatograms are shown representing the detection peaks of the substrate and product(s). Unfortunately, a computer image was not available.

Fig 12: 2-D Molecular representation of ω-2 hydroxylated C16 TMA BR with a total molecular weight of 380.447 Da. The positive charge of the N-terminus is neutralized by the bromide ion to yield a neutral molecule.

The R47E reaction sample that is injected yields many peaks that can be further zoomed into to analyze the peak separation at that specific retention time. In figure 13, there are 5 rows of chromatograms showing the detected peaks representing the substrate and product of one single injection of the R47E reaction. These five rows of graphs are analyzed for a better understanding of the formed peaks with respect to their retention times and base m/z values to decipher whether the peak represents a product. Ultimately, the detection of product is paramount due to the three shunt pathways that render the enzyme back to steady state without any conversion of the substrate towards the hydroxylated product.

In figure 13, the x-axis represents the retention time in minutes that is valid for all of the rows of the graphs and the y-axis represents the relative abundance that is separately given for each row. On the right hand side, the letters NL followed by a number value e.g NL: 7.35E6 represents the intensity of the peaks detected thus each row of chromatogram looks different. A low NL value represents a low intensity of the peak, and a high NL value represents a high intensity. The first row represents all of the peaks detected within the sample that is further analyzed by the

0

50

100

150

200

250

Pl 2 E7 Pl 6 A4 Pl 8 G6 Pl 10 H11 Pl 11 H11 Pl 12 F3 Pl 19 G5

Mut

ant/

R47

E S

lope

Rat

io (

%)

Uncoupling Forecast Compared to R47E in Percentage Library Pl 2-19

H2O2 Production

NADPH Consumption


22

zooming in on the detected peaks and presented in the following rows of chromatograms. The second row is the zoomed in peak with the retention time of 8.48 minutes that represents the substrate because of the m/z value of 283.50-284.50 shown on the right hand side under the intensity value of NL 3.52E4. This value indicates that it is a low intensity peak and that the majority of the substrate is converted to product indicating promising efficiency towards product formation, which is why in the following chromatograms little trace of the substrate peak is detected at this retention time. In the third row, the retention time of 6.52 minutes is zoomed in and this represents the hydroxylated product (addition of OH group) based on the m/z value of 299.50-300.50 that is given on the right hand side under the intensity value of NL: 8.81E5. This high peak intensity value compared to the other NL values given in the other rows of chromatograms indicates the high amount of product formation. In the fourth row, the peak with retention time 5.94 minutes is zoomed in and based upon the m/z value of 282.50- 283.50 shown on the right, this also indicates the substrate. The 1-m/z value loss from the base molecular weight of 283.50-284.50 Da is potentially due to the dissociation of a hydrogen atom along with the bromide ion. Another peak is detected at 5.96 minutes shown by the chromatograph on the fifth row. In regard to the NL value of 7.34E4, the m/z value of the detected peak is 315.50-316.50. This m/z value indicates a double hydroxylation in which the hydroxylated product potentially re–entered the active site to be hydroxylated again at a different position yielding two OH groups at two positions of hydroxylation. When this peak was further analyzed by its mass spectrum the intensity of this peak was given by the value NL: 6.92E4 at retention time 5.98 minutes (See Figure 14). In the mass spectrum, two strong peaks at 283.1667 and 316.333 m/z confirm that at the retention times of 5.94-5.98 there are two peaks that represent the substrate and a possible double hydroxylated product respectively. This specific phenomenon would have to be re-evaluated with an optimized method to prove its feasibility. However, it is clear that the substrate peak at retention time 8.48 minutes is barely visible in the other chromatograms, indicating the fully utilized substrate by the enzyme to form product that is detectable using this developed method. Table 4 represents the mass dissociation of hydroxylated C16 TMA for the clarification of the hydroxylation mechanism as well as the m/z values presented in figures 13 and 14.

Fig 13: LC-MS results of the detected peaks. The chromatogram given in the first row indicates all the detected peaks in one R47E reaction sample. Followed by the further analysis of these detected peaks in order to determine product formation that can be detected by the developed LC-MS method. The retention time of 8.48 minutes in the chromatogram of the second row represents the substrate due to the m/z value given on the right hand side. This peak is not seen in other rows of chromatograms thus indicating the conversion of the majority of the


23

substrate to product. Product is strongly indicated by the retention time of 6.52 minutes at peak intensity NL: 8.81E5 with the base peak m/z value of 299.50-300.50.

Fig 14: Mass spectrum of the peak at retention time 5.98 to show the order of dissociation of the product detected and for the clarification of whether double hydroxylation was feasible in regard to base peak m/z value of 315.50-316.50.

TABLE 4: MASS DISSOCIATION OF HYDROXYLATED C16 TMA BR Compound Molecular Weight (m/z) Explanation N(CH3)3 (CH2)15CH3 BR (C16 TMA BR)

364.45

1st dissociated group is Br 79-81 N(CH3)3 (CH2)15CH3 283.5-284.5 Hydroxylation is catalyzed by P450

BM3 thus addition of OH group to ω-3, ω-2, and/or ω-1 positions

N(CH3)3(CH2)16OH 299.5-300.5 Hydroxylated product of 16-Hydroxy-N,N,N-trimethyl-1-hexadecanaminium bromide


24

5.4. BCA ASSAY The standards of the BCA assay kit provided by the manufacturer yielded the following calibration curve, which was used for total soluble protein content determination.

Fig 15: The linear calibration curve of the BCA standards yielding a regression of 0.9938 and a curve equation for the calculation of protein content within the CFE.

From the linear calibration curve (Figure 15) and its corresponding equation (Eq.1), the average absorbance values of each of the samples were used to calculate (Eq. 2) for the overall soluble protein concentration within the CFE, taking the dilution factors into account. The overview for the total soluble protein content for each variant is presented in figure 16.

y = ax+ b Eq.1 x =y− ba Eq.2

Fig 16: The total soluble protein in the CFE calculated with Eq. 2 for each variant in µg/ml and their respective standard deviation.

y = 0.001x + 0.1522 R² = 0.9938

0.000

0.500

1.000

1.500

2.000

2.500

0 500 1000 1500 2000

Abs

orba

nce

562

nm

BSA Conc (μg/ml)

BSA Calibration Curve

00

1,000

2,000

3,000

4,000

5,000

6,000

7,000

8,000

9,000

10,000

P19

G5

P12

F3

P11

H11

P10

H11

P8 G

6

P6 A

4

P2 E

7

R47

E

WT

Tota

l Sol

uble

Pro

tein

(μ

g/m

l)

Variants

Total Soluble Protein in CFE of Chosen Variants


25

5.5. CO-DIFFERENCE SPECTRUM ASSAY The CO difference spectrum assay, as mentioned in section Materials and Methods 4.5, is applied to determine the amount of P450 present in the expressed culture. The combination of BCA assay and CO-difference assay designates the overall protein content and P450 protein content respectively. This shows the different levels of overall protein content within the expressed variant culture and how much of that protein content is P450 and thus a hypothesis on the activity towards product formation of a certain variant can be stated. In Figure 17 the absorbance curve of the consecutive reduction and gassing of the CFE in which the carbon monoxide binds to the heme porphyrin yields the active form of the enzyme with its characteristic absorbance at 450nm. Figure 18 presents the calculated P450 BM3 content in the CFE of the 7 chosen variants with the support of Table 5 presenting the absorbance values measured, the respectively calculated P450 BM3 concentration of each variant, and the percentage of P450 BM3 concentration in relation to total soluble protein content within the expressed culture.

Fig 17: Inactive form of the P450 has an absorbance of 420nm. From the curves it is apparent that with each gassing and reduction shows an absorbance spectrum that gradually shifts from 420 to 450nm. The results shown here are the spectrum of R47E as a control unit.

Absorbance of the highest peak of 450 nm as seen in figure 17 and the plateau line at 490 nm is noted and calculated by the Lambert-Beer equation:

𝐶!!"# ! 𝐴450 − 𝐴490

𝜀 ∗ 𝑙∗ 𝐹𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛 ∗ MW P450 BM3 Eq. 3

% 𝑜𝑓 𝐶𝑌𝑃450 =𝐶𝑜𝑛𝑐 𝑜𝑓 𝑃450

𝑇𝑜𝑡𝑎𝑙 𝑆𝑜𝑙𝑢𝑏𝑙𝑒 𝑃𝑟𝑜𝑡𝑒𝑖𝑛 (𝐶𝐹𝐸) Eq .4

-1

-0.5

0

0.5

1

1.5

2

2.5

370 390 410 430 450 470 490

Abs

orba

nce

Wavelength (nm)

P450 BM3-R47E CO Difference Absorbance Curves

reduced and gassed

reduced and gassed2

reduced and gassed3

reduced and gassed4


26

TABLE 5: ABSORBANCE VALUES AND P450 BM3 CONCENTRATIONS MEASURED BY CO DIFFERENCE SPECTRUM ASSAY

Mutants New Library A450 A490

P450 BM3 conc (mM)

P450 BM3 conc (μg/ml)

% of CYP450 to Tot Soluble Protein

P19 G5 -‐0,0020 -‐0,0321 0,0033 393,7 5,5 P12 F3 0,0346 -‐0,0085 0,0047 563,1 9,0 P11 H11 0,0077 -‐0,0215 0,0032 381,0 4,5 P10 H11 0,0657 0,0015 0,0070 838,9 12,0 P8 G6 0,0221 -‐0,0129 0,0038 457,3 6,4 P6 A6 0,0732 -‐0,0229 0,0106 1257,1 16,5 P2 E7 0,0523 0,0165 0,0039 468,0 6,6 R47E 0,0102 -‐0,0267 0,0040 481,9 7,1 WT -‐0,0313 -‐0,0238 0,0008 98,3 1,4

Fig 18: Concentration of P450 BM3 in the CFE of each variant is calculated by the Lambert-Beer formula (Eq. 3)

0

200

400

600

800

1,000

1,200

1,400

P19

G5

P12

F3

P11

H11

P10

H11

P8 G

6

P6 A

4

P2 E

7

R47

E

WT

P45

0 B

M3

Con

cent

rati

on (µ

g/m

l)

Variants

P450-BM3 Protein Concentrations of the Expressed Mutant Cultures


27

Fig 19: The ratios between the expression of all soluble protein within the host cell and the expression of P450 BM3. The total soluble protein content within the expressed culture and the expression of P450 BM3 determined by the CO difference spectrum assay are compared to give an overview of the level of expression within the host cell. Percentage of P450 BM3 to total soluble protein was calculated (Eq. 4) and presented in table 5.

5.6. GENE SEQUENCING The sequence results were aligned in comparison to the P450 BM3 wild type to determine the amino acid substation at the base pair position. As stated in 2.2.1 and 3.2, the four key amino acid residues in the active site were saturated and the amino acid substitution was determined by the analysis of their sequence shown in figures 20 and 21. Nucleotide base pair positions 201, 213, 321, and 624 designate the saturated amino acids R47, Y51, F87, and L188 respectively. The colors black, green, red, and blue in the sequence alignment of figure 20 and 21represent the nucleotides G, A, T, and C respectively. The resulting amino acid substitutions at four key amino acid residues saturated by the OmniChange method is presented in a full overview in table 6.

Fig 20: Sequence alignment of the chosen variants in comparison to that of P450 BM3 WT at position R47 with GAN codon saturation and at position Y51 with NYN codon saturation. The ruler indicates nucleotide base position of the respective gene sequences.

0

1,000

2,000

3,000

4,000

5,000

6,000

7,000

8,000

9,000

P19

G5

P12

F3

P11

H11

P10

H11

P8 G

6

P6 A

4

P2 E

7

R47

E

WT

Pro

tein

Con

cent

rati

on (µ

g/m

l)

Variants

Ratio of Total Soluble- and P450 BM3 Protein Content

BCA-Tot. Sol. Protein Conc

P450 conc


28

Fig 21: Sequence alignment of the chosen variants in comparison to that of P450 BM3 WT at position F87 with GNN codon saturation and at position L188 with NNK codon saturation. The ruler indicates the nucleotide base position of the respective gene sequences.

TABLE 6: AMINO ACID SUBSTITUTIONS RESULTING FROM THE FOCUSED DIRECTED MUTAGENESIS R47 Y51 F87 L188 P450 BM3 WT CGT- Arg TAT-Tyr TTT-Phe CTG -Leu Mutants ê R47-GAN Y51-NYN F87-GNN L188-NNK Pl 2 E7 CGT-> GAC

(Arg -> Asp) TAT-> TTG (Tyr-> Leu)

TTT-> GTG (Phe- Val)

CTG-> GCT (Leu-> Ala)

Pl 6 A4 CGT-> GAG (Arg-> Glu)

TAT-TTT (Tyr-> Phe)

TTT-> GGT (Phe-> Gly)

CTG-> GGG (Leu-> Gly)

Pl 8 G6 CGT-> GAT (Arg-> Asp)

TAT-> TTG (Tyr-> Leu)

TTT-> GTG (Phe-> Val)

CTG-> GGG (Leu-> Gly)

Pl 10 H11 CGT-> GAG (Arg-> Glu)

TAT-> ATG (Tyr-> Met)

TTT-> GTT (Phe-> Val)

CTG-> GAG (Leu-> Glu)

Pl 11 H11 CGT-> GAT (Arg-> Asp)

TAT-> ACG (Tyr-> Thr)

TTT-> GTG (Phe-> Val)

CTG-> GAG (Leu-> Gly)

Pl 12 F3 CGT-> GAA (Arg-> Glu)

TAT-> TTC (Tyr-> Phe)

TTT-> GTC (Phe-> Val)

CTG-> CAG (Leu-> Gln)

Pl 19 G5 CGT-> GAT (Arg-> Asp)

TAT-> TCA (Tyr- > Ser)

TTT-> GTC (Phe-> Val)

CTG-> TGT (Leu-> Cys)

6. DISCUSSION AND CONCLUSION The top 7 best performing variants in comparison to R47E are from Pl 2 E7, Pl 6 A4, Pl 10 H11, P11 H11, Pl 12 F3, and Pl 19 G5. These variants show a high NADPH consumption and low hydrogen peroxide formation, which indicates a coupling efficiency towards product. However, the NADPH consumption assay and the hydrogen peroxide assay does not specifically detect product formation thus these are indirect assays that indicate the variants’ coupling efficiency towards product formation. The statistical analysis of the selection of these variants were not applicable and were not proposed because all of the approximately 2,000 generated variants were screened repeatedly to ensure the consistency of the acquired data on their NADPH consumption and the production rate of hydrogen peroxide to narrow the selection of variants to at most the top 10 best performing mutants based on their rates. Screenings of the same plate of mutants from the master plate were done more than three times due to the high standard deviation given by the resulting data or data showing no activity, which leads to the conclusion that mistakes in the culturing procedure, was made and thus the procedure was repeated. This high deviating data was discarded and those that were consistent with each screening were kept for this report. The slope of the NADPH consumption rates for all of the 7 mutants range between ΔA/Δt of 0.00114 to 0.00240. The lowest slope results from Pl 2 E7 and the highest from Pl 11 H11. The R47E has a slope of 0.00116. When compared with the lowest performing mutant from PL 2 E7, only a difference 0.00002 was seen in activity indicating it is on par with R47E. However, this mutant and those that have very small difference in slope compared to R47E e.g. Pl 6 A4, were carried on to further analysis due to their NADPH consumption trend (Appendix F.1 Figure 13B) and their low yield in hydrogen peroxide. The lowest ratio percentage of NADPH consumption and hydrogen peroxide production compared to R47E resulted from the variant Pl 8 G6 at 30% indicated a high uncoupling forecast towards product formation. The standard deviations resulting from the hydrogen


29

peroxide assay was not avoidable despite the consecutive screenings to ensure no handling mistakes were affecting the standard deviation, however, due to a minor technical malfunction of the detector of the TECAN Infinite [as was later notified] was the main problem in high standard deviations.

In order to confirm the coupling efficiency of the top 7 best performing variants, product detection and quantification of the detected product must be performed. Due to time limitations for the optimization of the developed method, only with the unoptimized method, it was confirmed that the substrate and product was detectable. However, because this method was not optimized, the peaks given were a rough determination of product and substrate within one reaction (injection sample) of the control unit of R47E. From this sample, it was determined that the established detection of substrate and product was detected at peaks with retention times 8.48 minutes and 6.52 minutes respectively (Figure 13). From the given 5 rows of chromatograms representing one injection sample of the reaction R47E, the peak at 8.48 minutes is not visible in the other rows, therefore, it is safe to conclude a majority of the substrate is utilized in conversion towards the hydroxylated product that is detected at the retention time of 6.52 minutes. There was confusion regarding the fourth and fifth row chromatograms in which the peak at 5.94 minutes presented in the fourth chromatogram indicates substrate due to the given m/z value of 282.50-283.50 and in the fifth chromatogram a peak at 5.96 minutes indicates a doubly hydoxylated product due to the given m/z value of 315.50-316.50. Only when this retention time is further analyzed with a mass spectrum chromatogram (Figure 14) at 5.98 minutes, it shows two prominent spikes at m/z value 283.1667 and m/z value 316.3333 suggesting the possible overlap of peaks for the detection of both the substrate and the doubly hydroxylated product. Consequently, the investigation of this phenomenon requires an optimized LC-MS method in which defined peaks and retention times exclude the speculation of overlapping peaks that represent more than one compound. From the first three chromatograms, it can be concluded with a relative level of confidence that with the developed method the substrate and product are detectable but the detection method requires further optimization. The detection of product formation correlates to the concentration of P450 BM3 measured in the flask-expressed culture to present a relationship in the amount of product formed that is reliant on the P450 BM3 concentration measured in the expressed culture. The concentrations compared to the overall soluble protein content in the culture was much higher than that of P450 BM3 as expected, however, the concentrations of P450 BM3 were considerably low: the majority not exceeding 16%. This was speculated as to whether the high OD measurement at 600 nm yielding 1.3 could affect P450 expression whereas in the protocol the range must be kept at 0.8-1.0. Thus a possible over growth of culture could have lead to decreased expression of P450 BM3 despite the addition of the protein inducing agents: ALA, IPTG and thiamine.

The 7 top best performing mutants are generated with the OmniChange with the selection of saturation sites based on the active site amino acid residues of P450 BM3 to yield an enhanced enzyme with better catalytic properties. In order to define the amino acid substitutions performed by the OmniChange method, gene sequencing is necessary. In all of the selected mutants, the saturation site of R47 shows the predominant substitution of the arginine to glutamic acid or to aspartic acid, which is logical due to the GAN codon usage in the PTO primers used, to cause the substitution for the positive arginine to be substituted by a negative charged amino acid. Because the R47 facilitates the recognition of substrate and stabilizing the substrate upon entry and binding, the negative charged glutamate or aspartate substitution allows for the positively charged C16 TMA Br or alkyl trimethylammonium compounds to be recognized as a substrate for the variants but without the loss of catalytic activity towards its usual fatty acid substrates (6). The saturation site of Y51 in all of the 7 mutants have a variety of amino acids substituted instead of a predominant preference, these amino acids that have been substitute are leucine, phenylalanine, methionine, threonine, and serine. Y51 is a supporting amino acid residue in which it carries out stabilizing interactions with the substrate. The substrate-binding pocket is very hydrophobic thus the interesting aspects in the result of these substituted amino acids are the threonine and serine substitutions because they are polar amino acids that contain OH groups in contrast to leucine, phenylalanine, and methionine that are hydrophobic. The saturation site of F87 is predominantly substituted by the valine amino acid for 6 out of the 7 variants. F87 residue in the active site also is a supporting residue that is important in the substrate recognition, it forms interactions with the heme plane, and it is responsible for the regioselectivity of substrate oxidation (1,2). The substitution of valine is peculiar and interesting because compared to phenylalanine, it is a much smaller amino acid, therefore, this substitution is speculated to be involved in creating more space within the substrate-binding pocket for creating a lipophilic environment in which the regioselectivity and oxidation is facilitated. The saturation site of L188 shows a variety of amino acid substitutions in all of the 7 variants: showing no preference for a certain amino acid. These substitutions are alanine, glycine, glutamate, glutamine, and cysteine and all have different properties. L188 residue in the active site is important in substrate chain length selectivity thus for future prospects, it


30

would be interesting to focus on the catalytic activity of P450 BM3 with these amino acid substitutions implemented to show hydroxylation on different carbon chain length substrates (29).

From the generated 2,000 mutants, the selected top 7 best performing mutants show not only increased catalytic activity to that of R47E based on consecutive screenings of NADPH consumption and hydrogen peroxide assay, but also indicate promising potential towards coupling of product formation without the hindrance of the three shunt pathways in the catalytic cycle. In order to fully quantify which of the top 7 variants yield the most product, optimization of the LC-MS method and a coupled enzyme reaction system (biocatalysis) must be performed. The quantification of product by means of biocatalysis and LC-MS will also give insight on the correlation to P450 BM3 concentration determined by the CO difference spectrum assay and the amount of product detected. Those that yield the most product based on the analysis of LC-MS will narrow down the top 7 best performing to only one best performing variant. Recommended further research concerning this project is the kinetic characterization and whole genome sequencing of the best performing variant for official establishment of the best mutant than that of the recognized R47E. .

The prospects for the full characterization of the best performing variant and its establishment will prove very useful in many areas of science ranging from pharmaceutical to environmental processes. For example, engineering enzymes to carry out processes that are regularly done by chemical catalysts benefits the sustainability of synthesis in organic chemistry (15). Especially the catalysis of detergents and surfactants is fundamentally valuable for the detoxification of pollutants in the environment (7). The profound advancement of protein engineering benefits all sectors and inspires more sustainable and conscious processes within the industry.

7. ACKNOWLEDGEMENTS

I would like to thank everyone that has helped me throughout my thesis and internship. My gratitude goes especially to Prof. Dr. Ulrich Schwaneberg and of course to my supervisors Dr. Alan Mertens and Yu Ji for giving me the opportunity to collaborate with you and most importantly to learn from you.

Dr. Alan Mertens, thank you for teaching me about the fundamentals of P450 BM3 in our weekly meetings and answering all the questions I had even when they may have been futile. Also thank you for entrusting me with this project and being critical of my work. This has helped me to persevere and do better.

Yu Ji, thank you for your guidance during our lab hours so that I performed the experiments correctly and understood the underlying theory. I am grateful for your support, knowledge and foremost your guidance. It has been a pleasure to work with you and to learn from you.

I would also thank my internship coordinator Dr. Ralph Bax, and Dr. Maarten Vd Velden for your support and feedback during my internship so that everything went smoothly and accordingly for my graduation.

Furthermore, I’m grateful for the research team at RWTH for always being ready to help when I needed, and of course the lab technicians for all your maintenance in making sure everything is running perfectly.

Finally, it has been a great experience to work with all you at the RWTH Institute for Biotechnology!


31

8. REFERENCES 1. Oliver CF. The Role of Active Site Residues in Cytochrome P450 BM3 from Bacillus megaterium.

Leicaster: ProQuest LLC; 1998.

2. Munro AW, Leys DG, McLean KJ, Marshall KR, Ost TWB, Daff S, et al. P450 BM3: the very model of a modern flavocytochrome. Trends Biochem Sci [Internet]. 2002 May 1;27(5):250–7. Available from: http://www.sciencedirect.com/science/article/pii/S0968000402020868

3. Whitehouse CJC, Bell SG, Wong L-L. P450BM3 (CYP102A1): connecting the dots. Chem Soc Rev [Internet]. The Royal Society of Chemistry; 2012;41(3):1218–60. Available from: http://dx.doi.org/10.1039/C1CS15192D

4. Lamb DC, Waterman MR. Unusual properties of the cytochrome P450 superfamily. Philos Trans R Soc B Biol Sci [Internet]. 2013 Jan 6;368(1612). Available from: http://rstb.royalsocietypublishing.org/content/368/1612/20120434.abstract

5. Dennig A, Shivange A V., Marienhagen J, Schwaneberg U. Omnichange: The sequence independent method for simultaneous site-saturation of five codons. PLoS One. 2011;

6. Oliver CF, Modi S, Primrose WU, Lian LY, Roberts GC. Engineering the substrate specificity of Bacillus megaterium cytochrome P-450 BM3: hydroxylation of alkyl trimethylammonium compounds. Biochem J. 1997;327 ( Pt 2:537–44.

7. Lara-Martín PA, Li X, Bopp RF, Brownawell BJ. Occurrence of Alkyltrimethylammonium Compounds in Urban Estuarine Sediments: Behentrimonium As a New Emerging Contaminant. Environ Sci Technol [Internet]. American Chemical Society; 2010 Oct 1;44(19):7569–75. Available from: http://dx.doi.org/10.1021/es101169a

8. Munro AW, Girvan HM, McLean KJ. Variations on a (t)heme-novel mechanisms{,} redox partners and catalytic functions in the cytochrome P450 superfamily. Nat Prod Rep [Internet]. The Royal Society of Chemistry; 2007;24(3):585–609. Available from: http://dx.doi.org/10.1039/B604190F

9. Gonzalez FJ. The molecular biology of cytochrome P450s. Pharmacol Rev [Internet]. 1988 Dec 1;40(4):243–88. Available from: http://pharmrev.aspetjournals.org/content/40/4/243.short

10. Podust LM, Sherman DH. Diversity of P450 Enzymes in the Biosynthesis of Natural Products. Nat Prod Rep [Internet]. 2012 Oct 12;29(10):1251–66. Available from: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3454455/

11. Nelson DR, Koymans L, Kamataki T, Stegeman JJ, Feyereisen R, Waxman DJ, et al. P450 superfamily: update on new sequences, gene mapping, accession numbers and nomenclature. Pharmacogenet Genomics [Internet]. 1996;6(1). Available from: http://journals.lww.com/jpharmacogenetics/Fulltext/1996/02000/P450_superfamily__update_on_new_sequences,_gene.2.aspx

12. Nelson DR. The cytochrome p450 homepage. Hum Genomics [Internet]. 2009;4(1):59–65. Available from: http://www.humgenomics.com/content/4/1/59

13. Denisov IG, Makris TM, Sligar SG, Schlichting I. Structure and Chemistry of Cytochrome P450. Chem Rev [Internet]. American Chemical Society; 2005 Jun 1;105(6):2253–78. Available from: http://dx.doi.org/10.1021/cr0307143

14. Unno M, Matsui T, Ikeda-Saito M. Structure and catalytic mechanism of heme oxygenase. Nat Prod Rep [Internet]. The Royal Society of Chemistry; 2007;24(3):553–70. Available from: http://dx.doi.org/10.1039/B604180A

15. Warman AJ, Roitel O, Neeli R, Girvan HM, Seward HE, Murray SA, et al. Flavocytochrome P450


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BM3: an update on structure and mechanism of a biotechnologically important enzyme. Biochem Soc Trans [Internet]. 2005 Aug 1;33(4):747 LP-753. Available from: http://www.biochemsoctrans.org/content/33/4/747.abstract

16. Sevrioukova IF, Li H, Zhang H, Peterson JA, Poulos TL. Structure of a cytochrome P450–redox partner electron-transfer complex. Proc Natl Acad Sci [Internet]. 1999 Mar 2;96(5):1863–8. Available from: http://www.pnas.org/content/96/5/1863.abstract

17. Dennig A, Marienhagen J, Ruff AJ o??lle, Schwaneberg U. OmniChange: simultaneous site saturation of up to five codons. Methods Mol Biol. 2014;

18. Noble MA, Miles CS, Chapman SK, Lysek DA, MacKay AC, Reid GA, et al. Roles of key active-site residues in flavocytochrome P450 BM3. Biochem J [Internet]. 1999 Apr 15;339(Pt 2):371–9. Available from: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1220167/

19. Li QS, Schwaneberg U, Fischer P, Schmid RD. Directed evolution of the fatty-acid hydroxylase P450 BM-3 into an indole-hydroxylating catalyst. Chemistry. 2000;6(9):1531–6.

20. Tsai PC, Ding WH. Determination of alkyltrimethylammonium surfactants in hair conditioners and fabric softeners by gas chromatography-mass spectrometry with electron-impact and chemical ionization. In: Journal of Chromatography A. 2004.

21. Li X, Brownawell BJ. Analysis of quaternary ammonium compounds in estuarine sediments by LC-ToF-MS: Very high positive mass defects of alkylamine ions as powerful diagnostic tools for identification and structural elucidation. Anal Chem. 2009;81(19):7926–35.

22. Wong TS. Sensitive Assay for Laboratory Evolution of Hydroxylases toward Aromatic and Heterocyclic Compounds. J Biomol Screen [Internet]. 2005;10(3):246–52. Available from: http://jbx.sagepub.com/cgi/doi/10.1177/1087057104273336

23. Veitch NC. Horseradish peroxidase: A modern view of a classic enzyme. Phytochemistry. 2004;65(3):249–59.

24. Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, et al. Measurement of protein using bicinchoninic acid. Anal Biochem [Internet]. 1985;150(1):76–85. Available from: http://www.sciencedirect.com/science/article/pii/0003269785904427

25. Guengerich FP, Martin M V, Sohl CD, Cheng Q. Measurement of cytochrome P450 and NADPH–cytochrome P450 reductase. Nat Protoc [Internet]. 2009 Aug 6;4(9):10.1038/nprot.2009.121. Available from: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3843963/

26. Tabor S, Richardson CC. DNA sequence analysis with a modified bacteriophage T7 DNA polymerase. Proc Natl Acad Sci U S A [Internet]. 1987;84(14):4767–71. Available from: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=305186&tool=pmcentrez&rendertype=abstract

27. Zhu B. Bacteriophage T7 DNA polymerase - sequenase. Front Microbiol [Internet]. 2014;5(April):181. Available from: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=3997047&tool=pmcentrez&rendertype=abstract

28. Tabor S, Richardson CC. Selective inactivation of the exonuclease activity of bacteriophage T7 DNA polymerase by in vitro mutagenesis. J Biol Chem [Internet]. 1989;264(11):6447–58. Available from: http://www.ncbi.nlm.nih.gov/pubmed/2703498

29. Jang H-H, Shin S-M, Ma SH, Lee G-Y, Joung YH, Yun C-H. Role of Leu188 in the Fatty Acid Hydroxylase Activity of CYP102A1 from Bacillus megaterium. J Mol Catal B Enzym [Internet]. 2016 Nov;133:35–42. Available from: http://www.sciencedirect.com/science/article/pii/S1381117716301333


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9. LIST OF FIGURES Fig 1: The general catalytic cycle of cytochrome P450 showing the redox reactions of the heme porphyrin in relation

to its redox partner and substrate. Within the cascade, three alternative pathways called shunt pathways are shown to prove possible uncoupling scenarios in regard to enzyme-substrate complex (13). ................................... 7

Fig 2: Tertiary structure of the P450 BM3 protein. The heme domain is shown here in blue in which the heme plane in the active site is shown in red. The FAD domain is presented in green and the cofactor FMN is presented by yellow. 458 and 479 are the last visible residues in the heme domain and FAD domain, respectively (16). This image represents the monomer and thus the single polypeptide chain that make up the reductase and the heme domain however, the quaternary structure of this protein is comprised of two monomers thus having two substrate entry sites. The positioning of the heme plane in this figure is of importance considering relation of the entry site of the substrate in proximity to the heme. ...................................................................................................... 8

Fig 3: In section A, the upper molecule shows the natural nucleotide backbone of DNA, which is connected via phosphodiester bonds. The lower molecule is the phosphorothiolated DNA backbone in which the sulfur atom is indicated by an asterisk. In section B, the chemical cleavage of phosphorothiolated nucleotides with iodine and ethanol is shown under pH 9.0 (17). ............................................................................................................................... 9

Fig 4: Scheme of efficient oligonucleotide design using OmniChange method where the selection of the codon is targeted (3A). 12 nts are marked upstream the targeted codon for the designation of hybridization and complementary 12 PTO primer design (3B) The design of oligonucleotides with each of the 12 consecutive phosphorothiolated nts are introduced on the 5’ ends of each primer followed by the NNK codon and the rest 20 nts which are bonded by the usual phosphodiester bonds found in DNA and RNA yielding a total of 35 nts long forward primer in (3C) (17). ................................................................................................................................... 10

Fig 5: The Omnichange method scheme. 1) The amplification with PTO primers by means of standard PCR is done to simultaneously saturate up to 5 codons by using the NNK codon to generate genetic diversity in the selected positions. Followed by cleavage of the 12 PTO nts to create 5’ DNA overhangs in which correct hybridization is secured. Hybridization generates full-length plasmids with inserts ready for transformation into a vector (17). 11

Fig 6: Active site structure within the substrate binding pocket and surrounding significant amino acid residues. This structure was defined with the use of palmitoleic acid (shown in ‘color’) as substrate. As present, R47 and Y51 are positioned in the opening of the substrate access channel regulating the interaction with the substrate for stabilization upon binding and substrate induced heme iron spin shift. The position of F87 is in accord to its responsibility of shielding the terminal methyl end from any hydroxylation or oxidation. L181 being positioned in the middle of the substrate entrance channel is synergistic with its role of regulating the carbon chain length of substrate (1, 7). ................................................................................................................................................................ 12

Fig 7: Molecule shown is the substrate positively charged C16 trimethylammonium bromide hydroxylated in the positions ω-1, ω-2, and ω-3. P450 BM3 R47E variant at one position per reaction cycle hydroxylates the substrate. ........................................................................................................................................................................... 13

Fig 8: Kinetic constants correlating the wild type P450 BM3 with that of P450 BM3 R47E mutant. In the seventh column with the constant kcat/Km it is clear that the mutant activity on the substrates C12-C16 TMA give a higher turnover frequency when compared with the wild type (6). ............................................................................ 13

Fig 9: An example of the BCA Colorimetric assay on MTP with standards ranging from 20 to 2000 µg/ml on first three rows. At the bottom right hand is the samples in three dilutions ranging from 1:5, 1:8, and 1:10 done in triplicates. MTP is scanned by TECAN Sunrise spectrophotometer at 562 nm. ...................................................... 17


34

Fig 10: Slope of the NADPH consumption of the chosen variants regarding the NADPH Consumption assay. Pl 11 H11 and Pl 19 G5 yield the highest slope in comparison to that of R47E, showing a higher activity than that of the control group (R47E). ................................................................................................................................................ 20

Fig 11: The hydrogen peroxide formation in comparison to that of NADPH consumption ratios for each variant are given in percentage in regard to R47E as benchmark. ................................................................................................ 21

Fig 12: 2-D Molecular representation of ω-2 hydroxylated C16 TMA BR with a total molecular weight of 380.447 Da. The positive charge of the N-terminus is neutralized by the bromide ion to yield a neutral molecule. ........... 21

Fig 13: LC-MS results of the detected peaks. The chromatogram given in the first row indicates all the detected peaks in one R47E reaction sample. Followed by the further analysis of these detected peaks in order to determine product formation that can be detected by the developed LC-MS method. The retention time of 8.48 minutes in the chromatogram of the second row represents the substrate due to the m/z value given on the right hand side. This peak is not seen in other rows of chromatograms thus indicating the conversion of the majority of the substrate to product. Product is strongly indicated by the retention time of 6.52 minutes at peak intensity NL: 8.81E5 with the base peak m/z value of 299.50-300.50. į5672 ............................................................................ 22

Fig 14: Mass spectrum of the peak at retention time 5.98 to show the order of dissociation of the product detected and for the clarification of whether double hydroxylation was feasible in regard to base peak m/z value of 315.50-316.50. ................................................................................................................................................................ 23

Fig 15: The linear calibration curve of the BCA standards yielding a regression of 0.9938 and a curve equation for the calculation of protein content within the CFE. ............................................................................................................... 24

Fig 16: The total soluble protein in the CFE calculated with Eq. 2 for each variant in µg/ml and their respective standard deviation. ........................................................................................................................................................... 24

Fig 17: Inactive form of the P450 has an absorbance of 420nm. From the curves it is apparent that with each gassing and reduction shows an absorbance spectrum that gradually shifts from 420 to 450nm. The results shown here are the spectrum of R47E as a control unit. ................................................................................................................. 25

Fig 18: Concentration of P450 BM3 in the CFE of each variant is calculated by the Lambert-Beer formula (Eq. 3) ... 26

Fig 19: The ratios between the expression of all soluble protein within the host cell and the expression of P450 BM3. The total soluble protein content within the expressed culture and the expression of P450 BM3 determined by the CO difference spectrum assay are compared to give an overview of the level of expression within the host cell. Percentage of P450 BM3 to total soluble protein was calculated (Eq. 4) and presented in table 5. ............. 27

Fig 20: Sequence alignment of the chosen variants in comparison to that of P450 BM3 WT at position R47 with GAN codon saturation and at position Y51 with NYN codon saturation. The ruler indicates nucleotide base position of the respective gene sequences. ....................................................................................................................................... 27

Fig 21: Sequence alignment of the chosen variants in comparison to that of P450 BM3 WT at position F87 with GNN codon saturation and at position L188 with NNK codon saturation. The ruler indicates the nucleotide base position of the respective gene sequences. .................................................................................................................... 28


35

APPENDIX APPENDIX A: OMNICHANGE PROTOCOL OmniChange protocol is as followed on this publication: (17)

APPENDIX B: CULTURING AND HIGH THROUGHPUT SCREENING

B.1. PROTOCOL: PRECULTURE AND CULTURING OF MUTANTS FOR SCREENING Materials and Method

1. LB medium (200 ml)

Peptone 2 gr Yeast Extract 1 gr NaCl 2 gr Dissolve peptone, yeast extract and NaCl in deionized water and fill to final volume of solution to 200 ml. Autoclave the media.

2. TB Medium (1000 ml)

Solution A (800 ml) Peptone 12 gr Yeast Extract 24 gr Glycerol 4 ml or 4 gr Dissolve peptone, yeast extract and glycerol in deionized water. The final volume of the mixture should be 800 ml. Solution B (200 ml)

KH2PO4 2.31 gr K2HPO4 12.54 gr Dissolve KH2PO4 and K2HPO4 in deionized water and fill up to 200 ml

Autoclave solutions A and B separately and mix them under sterile conditions to prepare a final volume of 1L of TB media.

3. Trace Elements Solution (1L: 1000x concentrated)

CaCl2.2H2O 0.5 gr ZnSO4.7H2O 0.18 gr MnSO4.7H2O 0.10 gr Na2-EDTA 20.10 gr FeCl3.6H2O 16.70 gr CuSO4.6H2O 0.18 gr Dissolve all components in 1L of deionized water. Autoclave and filter under sterilized conditions. Store at 4 degrees Celsius. Add 1ml per 1L medium.

4. ALA (Aminolevulinic acid) (0.5 M: 1000x concentrated)

Dissolve 0.837 gr of ALA in 10 ml Milli-Q water. Filter with a 0.2 µm filter and store at -20 degrees Celsius. Add 1 ml per 1 L medium yielding a final concentration of 100 µM.

5. IPTG (Isopropyl-‐β-‐D-‐thiogalactopyranoside) (0.1 M: 1000x concentrated)

Dissolve 0.2383 gr IPTG in 10 ml Milli-Q water and filter with a 0.2 µm filter. Store at -20 degrees Celsius. Add 1 ml per 1 L medium yielding a final concentration of 100 µM.


36

6. Kanamycin (50 mg/ml ; 1000x concentrated)

Add 0.5 gr to 10 ml Milli-Q water. Filter using a 0.2 µm filter and store in 1 ml aliquots under -20 degrees Celsius. Add 1 ml per 1 L medium.

7. Phosphate Buffer (KPi-‐Buffer, pH 8.0; 100mM)

0.25 L of 1M K2HPO4 (43.545 gr to 250 ml dH2O) 0.1 L of 1 M KH2PO4 (13.609 gr to 100 ml dH2O) For 1 L of Phosphate buffer add 94 ml of 1 M K2HPO4 and 6 ml of 1 M KH2PO4 and fill to 1 liter with deionized water. Check the pH using pH meter.

Pre-culture in 96 well Flat Bottom MTP (STERILE CONDITIONS) (22)

1. Pipet in each well of the 96-well flat bottom MTP 150 µl LB medium that is enriched with Kanamycin (ratio of Kanamycin to medium is 1:1000)

2. Transfer 4 µl of each mutant colony from the 96 wells of the master plate flat bottom MTP using an Eppendorf 0.5-10 µl pipet.

3. Grow overnight in a microplate shaker at 37 degrees Celsius, 9090 rpm, and 70% humidity.

Culture in 96 deep-well plates (STERILE CONDITIONS)

4. Pipet 600 µl of TB medium enriched with IPTG and ALA solutions to each of the 96-deep well plates. 5. Transfer 4 µl of precultured colony from the flat bottom MTP to each well of the deep well plate. 6. Incubate for 24 hours in a microplate shaker at 30 degrees Celsius, 900 rpm and 70% Humidity 7. Transfer 400 µl of each harvested cultures to a V-bottom MTP using Eppendorf 20-200µl pipet (Transferring

doesn’t have to be in sterile conditions). 8. Centrifuge for 15 minutes at 4 degrees Celsius and 4000 rpm using MTP Eppendorf Centrifuge 5810R 9. Discard the supernatant using Eppendorf 20-200 µl pipet and store the cell pellets overnight in -20 degrees

Celsius.

Flask Expression Preculture (STERILE CONDITIONS) Inoculate 4 ml LB + 4 μl Kanamycin with 4 μl of a glycerol stock culture, or with a single colony from a fresh agar plate. Grow the culture overnight at 900 rpm, 37 degrees Celsius Culture (STERILE CONDITIONS)

1. Inoculate 50 ml TB medium + 50 µl Kanamycin with 500 µl of the overnight culture (1%) 2. Incubate the culture at 250 rpm, 30°C until OD600 reaches between 0.8 and 1.0 (Check approximately after 2

hours). 3. Supplement the culture with 50 µl ALA and 50 µl Thiamine. Induce expression with addition of 50 µl IPTG to

sample and control. 4. Expression incubation time is 20-24 hours at 30°C, 250 rpm.

Crude extract preparation (No sterile conditions but prepared in 4°C or on ice)

1. Transfer the cultures into falcon tubes (50 ml) and centrifuge for 10 min, 4000 rpm (Eppendorf Centrifuge 5810R) 4°C.

2. Discard the supernatant and wash the cells with 10 ml of PBS (pH 7.4) or KPi buffer (pH 7.5, 50 mM) once. Centrifuge as in step 1.


37

3. Cell lysis: Discard the supernatant and add 3 ml of lysozyme mix (5 g/L in phosphate buffer). Vortex the tube to bring the pellet into a homogeneous suspension. Incubate at 250 rpm, 37°C for 30 minutes. Sonicate (parameters: 40% of amplitude, 3x30 sec on ice). (Sonics Vibracell VCX-130)

4. Centrifugation: 13000 rpm (Eppendorf Centrifuge 5415R) 30 minutes, at 4°C. 5. The supernatant is the cell free extract (CFE). Store at 4°C

B.2. PROTOCOL: NADPH CONSUMPTION ASSAY MATERIALS DMSO NADPH (0.5 mM) Potassium Phosphate Buffer 100 mM, pH 8.0 (KPi Buffer) C16 TMA Br (1.250 mM solution in DMSO) 96-well Flat Bottom Microtiter Plate Lysozyme (5 g/L) In V-bottom MTP Expressed P450-BM3 variant cells MTP Shaker (37 degrees Celsius, Humidity 70%, 900 rpm) MTP Centrifuge (4000 rpm, 4 degrees Celsius) (Eppendorf Centrifuge 5810R) TECAN Sunrise Spectrophotometer (Filter 340 nm) PROCEDURE

1. Cell lysis a. Let plates defreeze for 20 minutes. Add 75 µl phosphate buffer to the 96 V-bottom MTP plates with

the expressed mutant cells and let stand for 15 minutes. b. Add 125 µl freshly prepared lysozyme solution in phosphate buffer to each well and mix well by

pipetting up and down. c. Incubate the plates for 1 h at 37 degrees Celsius in the MTP shaker. d. Gain the cell free extract by centrifugation for 25 minutes at 4000 rpm in MTP centrifuge

(Eppendorf Centrigufe 5801R) and proceed with the assay.

2. NADPH Consumption Assay a. Before beginning the assay, prepare NADPH solution according to the molarity and check for the OD

using a spectrophotometer at 340 nm. The OD should be between 0.4-0.5 A to yield a molarity of 0.6-0.8 mM NADPH using the extinction coefficient of NADPH at 6.22 M-1 cm-1. Keep solution always on ice.

b. Pipet scheme of Sample and Control plate:

TABLE 6: PIPET SCHEME OF NADPH CONSUMPTION ASSAY Compounds Sample Control Notes DMSO with Substrate 1.25 mM

10 µL (with substrate) 10 µl (Only DMSO) Final conc. 50 µM (diluted 1:25)

100 mM KPi Buffer 155 µL 155 µL CFE 35 µL 35 µL Mix well!

3. Measuring using TECAN Sunrise Spectrophotometer

a. Place the correct 340 and 410 nm filter. Set de absorbance at 340 nm. Make an end to end measurement without adding the NADPH by clicking START MEASUREMENT. After the end to end measurement, in the kinetics tab of the program, assign 70 cycles with 13 second intervals. After incubating the substrate with the P450 BM3 (mutant) enzyme for 5 minutes, the conversion of substrate is started by adding NADPH;


38

NADPH 0.5 mM 50 µL 50 µL Final Conc. 160µM (diluted 1:5)

Total 250 µL 250µL

b. Immediately after adding NADPH the reaction will begin and start the 70 cycle kinetic measurement by clicking START MEASUREMENT on the program. It will take 15 minutes per 70 cycles.

c. Once the 70 cycles are done, repeat step A and B for the control plate

B.3. PROTOCOL: HYDROGEN PEROXIDE ASSAY

A NADPH/AmplexRed™ HTS assay for P450 BM3 evolution (coupling efficiency) (Version 1.0 by Dr. Thomas Horn RWTH Institute for Biotechnology/Internal Protocol)

Principle

The reaction consists out of two half reactions: 1. NADPH depletion assay: The P450 monooxygenase consumes NADPH under the formation of a hydroxylated/epoxidated product and water. 2. Amplex Red™ assay: As an uncoupling product hydrogen peroxide is formed which then can further react with the Amplex Red™ dye and the horseradish peroxidase (HRP) forming resorufin. This assay is applied to the substrate C16 TMA BR instead of anisole but the principle is the same.

Procedure

1) HTS Assay (room temperature)

1. NADPH depletion assay

• Final volume of the 1st half reaction is 100 µl, use black flat bottom 96-well MTPs • Pipette in this order (volumes per well):

o 70-x µl phosphate buffer pH 7.5 o 10 µl enzyme solution (supernatant) o x µl substrate solution #


39

o Start reaction by adding 20 µl NADPH solution (stock solution 1 mM ≙ 7.4 mg in 10 ml phosphate buffer pH 7.5)

• Tecan M200/1000 settings: fluorescence Exc. 340/460 nm; intervals 30-60 sec, shake 2 sec before first measurement; time 5-10 min

• Stop reaction by adding 25 µl acetonitrile (20% v/v) • Incubate at RT for 5-10 min

2. Amplex Red™ assay

• Start 2nd half reaction by adding 125 µl HRP/Amplex Red™ stock solution (2 U HRP, 8 µM Amplex Red™ dissolved in phosphate buffer pH 7.5); final conc. 1 U HRP/4 µM Amplex Red™ dye

• Tecan M200/1000 settings: fluorescence Exc. 535/590 nm; intervals 30-60 sec, shake 2 sec before first measurement; time 30 min

Remarks:

• for NADPH depletion assay: make a DMSO control plate (no substrate) • for Amplex Red assay: DMSO plate not required • analyze the slope of both half reactions to search for improved variants • Aim is to search for variants with “high” NADPH consumption and “low” hydrogen peroxide formation


40

APPENDIX C: BCA ANALYSIS PROTOCOL

MATERIALS

Pierce BCA Protein Assay Kit (ThermoFisher Scientific, Catalog # 23225) Bovine Serum Albumin (BSA) Cell Free Extract Potassium phosphate buffer 100 mM, pH 8.0 (KPi Buffer) TECAN Sunrise Spectrophotometer PROCEDURE

1. The manual within the kit was followed accordingly with the following alterations. 2. Weigh roughly 20 mg of BSA and dissolve in 10 ml of KPi buffer. This is the stock solution of BSA. 3. Standard Curve Dilutions are as follows: Vial # Dilutions Concentration

(µg/ml)

A Stock solution of BSA (~20mg/10ml) 2000

B 750 µl of Vial A + 250 µl KPi 1500

C 500 µl of Vial A + 500 µl KPi 1000

D 500 µl of Vial B + 500 µL KPi 750

E 500 µl of Vial C + 500 µl KPi 500

F 500 µl of Vial E + 500 µl KPi 250

G1 500 µl of Vial F + 500 µl KPi 125

G2 100 µl of Vial F + 400 µl KPi 50

H 200 µl of Vial G2 + 200 µl KPi 25

I 500 µl KPi Buffer 0

4. Sample Dilutions are as follows: Vial # Dilutions Volume of CFE (µl) Volume of KPi

buffer (µl)

1 1:5 100 400

2 1:8 50 350

3 1:10 30 270


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APPENDIX D: CARBON MONOXIDE DIFFERENCE ASSAY PROTOCOL Materials

Potassium Phosphate Buffer 100 mM, pH 8.0 (KPi Buffer) Cell Free Extract Sodium dithionite (solid form) Carbon monoxide tank (Sigma Aldrich) Carbon monoxide fume hood chamber Plastic 1 ml cuvet Spectrophotometer (VARIAN Cary 50 Bio UV-Visible Spectrophotometer)

Procedure

1. To record zero, 1 ml of KPi buffer is added to the cuvet and measured. 2. 1 ml of CFE is added to cuvet to record baseline. 3. Spectrum of 1 ml CFE is recorded and named as “unreduced and ungassed.” 4. Very small amount (tip of spatula) of sodium dithionite is added to the cuvet containing only CFE as a reducing

agent. 5. Spectrum is recorded again and named as “reduced and ungassed.” 6. In the fume hood chamber containing CO tank is connected to a small needle with a pressure regulator and gas

flow. The needle is inserted to the cuvet containing 1 ml CFE and gassed for 30 seconds. Bubbling indicates the flow of CO into the cuvet.

7. Spectrum is directly recorded and named as “reduced and gassed.” 8. The bubbling/gassing step is repeated and named accordingly until the spectrum peak of 420 nm disappears

and a peak at 450 nm is formed. This indicates the porphyrin ring and CO binding complex and thus the folding of the protein is correct.

9. When a large peak at 450 nm is formed and a plateau line is visible after the 450 nm mark, the absorbance at 490 nm is formed to record the plateau value.

10. Using the Lamber-‐Beer formula and making alterations to calculate the concentration, the concentration of P450 in a sample can be determined. The molar extinction coefficient of the porphyrin ring is 91 mM-‐1 cm-‐1 and the cuvet pathway is 1 cm.


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APPENDIX E: LC-MS METHOD

Purification/Extraction

Controls: R47E

Empty vector (EV)

Wild type (WT)

1. Collect up to 0.5-‐1 ml samples from the NADPH consumption assay done in MTP plates; Extraction is done with the ratio of 1:1 addition of chloroform. Vortex briefly and then sonicate (Sonics Vibracell VCX-‐130) for 3 minutes at 40% amplitude. Centrifuge (Eppendorf Centrifuge 5415R) for 5 minutes at top speed. A two-‐phase system should be visible. Pipet the organich phase (upper portion) in a separate 1.5 ml eppendorf tube.

2. Repeat step 1 with chloroform again 3. Add the two extractions (organic phase) together. 4. Transfer the supernatant in to GC vials with inlets of maximum 200 μl. Label the vials accordingly with the correct

used compound for extraction (chloroform) 5. Samples are ready for injection.

Preperation of LC-MS Standard: 10 mg/ml and 1 mg/ml Column: C18, with temperature at 30 degrees Celsius. Mobile Phase Solvent A: 20:80 ratio acetonitrile to water with 1% acetic acid. Solvent B: 95:5 ratio acetonitrile to water with 10 mM ammonium acetate Method: The Gradient Elution Mode

Time (min) Flow rate (ml*min-1)

A% B% Curve

Initial 0,5 100 0 Initial 2.0 0.5 20 80 1 2.1 0.5 20 80 1 7.1 0.5 0 100 6 13.0 0.5 0 100 6 31.0 0.5 50 50 6 35.0 0.5 100 0 1

1. Gradient conditions are initiated at 100% A and maintained for 2 minutes. 2. The linear gradient is changed to 20% A and 80% B in 0.1 minutes and held for 5 minutes. 3. Linear gradient is changed to 100% B and held for 2 minutes. 4. Linear gradient is changed to 90% B and held for 6 minutes. 5. Linear gradient is changed to 50% B and maintained for 18 minutes before the column is equilibrated to initial

conditions.


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APPENDIX F: RAW DATA

F.1. NADPH CONSUMPTION ASSAY

Fig 10B: Trend of selected active mutants from plates 2-19 over the period of 70 cycles with each cycle of 13-second intervals. The trends of the most active mutants are present with respect to a common starting point and a steep decrease in the curve of NADPH utilization per reaction i.e. Pl 11 H11.

F.2 HYDROGEN PEROXIDE ASSAY

Fig 11B: Trend of peroxide production in selected active mutants from plates 2-19 over the period of 61 cycles with each cycle of 30-second intervals resulting to total measurement time of 30 minutes. The trends of the selected active mutants are present with respect to a common starting point and an increase in the fluorescence with time. Trend of this plate was measured to 540 seconds (9 minutes) because the peroxide production reached detection limit.

0.250

0.350

0.450

0.550

0.650

0.750

0.850

0 38

77

116

155

194

233

272

311

350

389

428

467

506

545

584

623

662

701

740

779

818

857

896

Abs

orba

nce

340

nm

Time (s)

Trend of Selected Active Mutants NADPH Consumption

Library Pl 2-19

Pl 2 E7 Pl 6 A4 Pl 8 G6 Pl 10 H11 Pl 11 H11 Pl 12 F3 Pl 19 G5 R47E WT EV

0

10000

20000

30000

40000

50000

60000

70000

0 30

60

90

120

150

180

210

240

270

300

330

360

390

420

450

480

510

540

Fluo

resc

ence

535

/590

nm

Time (s)

Trend of H2O2 Production of Selected Active Mutants Library Pl 2-19

Pl 2 E7-H2O2

Pl 6 A4-H2O2

Pl 8 G6-H2O2

Pl 10 H11-H2O2

Pl 11 H11-H2O2

Pl 12 F3-H2O2

Pl 19 G5-H2O2

R47E

WT


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mproving p450 bm3 towards conversion

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