Engineering the Enantioselectivity and Thermostability of a(�)-�-Lactamase from Microbacterium hydrocarbonoxydans forKinetic Resolution of Vince Lactam (2-Azabicyclo[2.2.1]hept-5-en-3-one)

Shuaihua Gao,a,b,c Shaozhou Zhu,a Rong Huang,a Hongxia Li,a Hao Wang,a Guojun Zhenga

aState Key Laboratory of Chemical Resources Engineering, Beijing University of Chemical Technology, Beijing,People's Republic of China

bDepartment of Chemistry, University of California, Berkeley, Berkeley, California, USAcCalifornia Institute for Quantitative Biosciences, University of California, Berkeley, Berkeley, California, USA

ABSTRACT To produce promising biocatalysts, natural enzymes often need to beengineered to increase their catalytic performance. In this study, the enantioselectiv-ity and thermostability of a (�)-�-lactamase from Microbacterium hydrocarbonoxy-dans as the catalyst in the kinetic resolution of Vince lactam (2-azabicyclo[2.2.1]hept-5-en-3-one) were improved. Enantiomerically pure (�)-Vince lactam is the keysynthon in the synthesis of antiviral drugs, such as carbovir and abacavir, which areused to fight against HIV and hepatitis B virus. The work was initialized by usingthe combinatorial active-site saturation test strategy to engineer the enantiose-lectivity of the enzyme. The approach resulted in two mutants, Val54Ser andVal54Leu, which catalyzed the hydrolysis of Vince lactam to give (�)-Vince lac-tam, with 99.2% (enantiomeric ratio [E] � 200) enantiomeric excess (ee) and99.5% ee (E � 200), respectively. To improve the thermostability of the enzyme,11 residues with high temperature factors (B-factors) calculated by B-FITTER orhigh root mean square fluctuation (RMSF) values from the molecular dynamicssimulation were selected. Six mutants with increased thermostability were ob-tained. Finally, the mutants generated with improved enantioselectivity and mu-tants evolved for enhanced thermostability were combined. Several variantsshowing (�)-selectivity (E value � 200) and improved thermostability were ob-served. These engineered enzymes are good candidates to serve as enantioselec-tive catalysts for the preparation of enantiomerically pure Vince lactam.

IMPORTANCE Enzymatic kinetic resolution of the racemic Vince lactam using (�)-�-lactamase is the most often utilized means of resolving the enantiomers for thepreparation of carbocyclic nucleoside compounds. The efficiency of the native en-zymes could be improved by using protein engineering methods, such as directedevolution and rational design. In our study, two properties (enantioselectivity andthermostability) of a �-lactamase identified from Microbacterium hydrocarbonoxydanswere tackled using a semirational design. The protein engineering was initialized bycombinatorial active-site saturation test to improve the enantioselectivity. At thesame time, two strategies were applied to identify mutation candidates to enhancethe thermostability based on calculations from both a static (B-FITTER based on thecrystal structure) and a dynamic (root mean square fluctuation [RMSF] values basedon molecular dynamics simulations) way. After combining the mutants, we success-fully obtained the final mutants showing better properties in both properties. Theengineered (�)-lactamase could be a candidate for the preparation of (�)-Vincelactam.

Received 17 August 2017 Accepted 29September 2017

Accepted manuscript posted online 20October 2017

Citation Gao S, Zhu S, Huang R, Li H, Wang H,Zheng G. 2018. Engineering theenantioselectivity and thermostability of a(+)-γ-lactamase from Microbacteriumhydrocarbonoxydans for kinetic resolution ofVince lactam (2-azabicyclo[2.2.1]hept-5-en-3-one). Appl Environ Microbiol 84:e01780-17.https://doi.org/10.1128/AEM.01780-17.

Editor Ning-Yi Zhou, Shanghai Jiao TongUniversity

Copyright © 2017 American Society forMicrobiology. All Rights Reserved.

Address correspondence to Guojun Zheng,zhenggj@mail.buct.edu.cn.

ENZYMOLOGY AND PROTEIN ENGINEERING

crossm

January 2018 Volume 84 Issue 1 e01780-17 aem.asm.org 1Applied and Environmental Microbiology

on July 28, 2020 by guesthttp://aem

.asm.org/

Dow

nloaded from

KEYWORDS enantioselectivity, �-lactamase, semirational design, thermostability,Vince lactam

Enzymes are excellent catalysts with a broad range of activities, such as the enan-tioselective hydrolysis of the Vince lactam (2-azabicyclo[2.2.1]hept-5-en-3-one) by

(�)-�-lactamases to give (�)-Vince lactam (1–6). The enantiopure (�)-Vince lactam is anindispensable building block for the preparation of antiviral agents, such as carbovirand abacavir (Fig. 1). The enantioselective resolution of Vince lactam using appropriateenzymes provides a straightforward and attractive method to access the chiral inter-mediate due to the excellent enantioselectivity and environmental friendliness (noheavy metals, such as titanium, are needed) (5–7). To date, several (�)-�-lactamaseshave been purified and biochemically characterized for their potential in the prepara-tion of enantiopure Vince lactam (6, 8–16). However, their applications have beenlimited because of unsatisfactory stereoselectivity, low activity, or insufficient thermo-stability.

To produce efficient biocatalysts, natural enzymes often need to be redesigned toenhance their catalytic performance (17). Enzyme engineering is the process by whichthe natural sequence of an enzyme is altered in favor of a particular property. A numberof unique and exclusive enzyme engineering strategies are currently employed tomodify biocatalysts, improving their performance for practical applications (17–23).These include various directed evolution techniques, rational/semirational design tech-niques, rational and computational designs, and more recently, the de novo design ofnovel enzymes. Properties, including thermostability, substrate spectrum, enzyme ac-tivity, and enantioselectivity, of some natural proteins could be improved efficientlybased on these strategies (24–28). The enantioselectivity of an esterase for the asym-metric hydrolysis of aryl prochiral diesters was controlled by introducing aromaticinteractions, which demonstrated that aromatic interaction is one of the origins ofenzyme enantioselectivity (29). In addition, the catalytic efficiency of a short-chaindehydrogenase/reductase was enhanced by reconstruction of the catalytic pocket andenzyme-substrate interactions. The resulting variants showed significantly improvedcatalytic efficiency (the kcat/Km value was 15-fold greater than that of the wild type)toward a series of prochiral ketones in some cases (30).

A �-lactamase from Microbacterium hydrocarbonoxydans (designated MhIHL) withhigh enzyme activity is the subject of the present study (16, 31). MhIHL is a smallenzyme with a molecular mass of 20 kDa. Although it shows �-lactamase activity, it isevolutionarily, structurally, and biochemically distinct from all known (�)-�-lactamases(6–9, 11, 14), i.e., it does not belong to the classic amidase family. It is assigned to theisochorismatase-like hydrolase (IHL) superfamily/cysteine hydrolase family, CDD classi-fication cd00431 (32). Like other IHLs, MhIHL folds into a typical �/�-fold with asix-stranded parallel �-sheet in the middle, flanked by three helices, and a single longhelix on both sides of the sheet (31). MhIHL has the conserved catalytic triad D13-K78-C111, with the C111 acting as the nucleophile. The proposed mechanism resembles themechanism proposed for other �/�-hydrolase enzymes, such as those from Acineto-bacter baumannii PncA26 (31). Structural analysis indicates that MhIHL lacks a loop inthe entrance to the binding pocket compared to other IHLs. We suggest that this openconformation of the active cavity in MhIHL would facilitate both the binding of thesubstrate and the release of the product molecule but simultaneously reduce thespecificity of the substrate, consistent with the comparable enzymatic kinetic param-eters of the two enantiomers catalyzed by MhIHL (31). This promiscuous enzymedisplays 10 times higher (�)-�-lactamase activity than the most active (�)-�-lactamasereported before (Table 1), making it a very good target for the preparation of enan-tiopure (�)-Vince lactam. A previous study showed that MhIHL could catalyze thehydrolysis of both enantiomers with a specific hydrolysis curve (31); thus, it is necessaryto improve its enantioselectivity before it can be considered for practical applications.Another property which needs to be tailored is its poor thermostability. Excellent

Gao et al. Applied and Environmental Microbiology

January 2018 Volume 84 Issue 1 e01780-17 aem.asm.org 2

on July 28, 2020 by guesthttp://aem

.asm.org/

Dow

nloaded from

thermostability is an attractive property of catalysts because it allows for long-termstorage and resilience under harsh conditions, thus reducing costs (33). Given theabove-described situation, our task was to engineer the enantioselectivity and ther-mostability of the protein and prevent the loss of its original activity as much aspossible. As such, the wild-type MhIHL was chosen as the starting template forengineering. Engineering of the enantioselectivity was initialized using the combina-torial active-site saturation test (CAST) strategy. Meanwhile, the engineering of ther-mostability was started based on the calculated thermostability-related factors. Finally,the point mutations of the best variants were combined to determine the overall bestmutants. The best two double mutants, Arg162Thr-Val54Leu and Glu95Lys-Val54Ser,both showed better enantioselectivity and thermostability. Even though a trade-off inactivity was observed for both of these mutants compared to other reported�-lactamases, the mutants still display the highest catalytic efficiency.

RESULTSScreening for improved enantioselectivity from the libraries. MhIHL can catalyze

the hydrolysis of both enantiomers of Vince lactam, but with apparently differentspecificities (31). Although it was possible to obtain the expected (�)-enantiomer insufficient enantiomeric excess (ee) and conversion ratio by controlling the reactionconditions properly (16), the protein was not suitable for practical application withoutoptimization. To shed light on the structure and molecular basis for engineering ofMhIHL, we first solved the X-ray structure of MhIHL in complex with substrates andelucidated the catalytic mechanism (31). The engineering process was illustrated in Fig.2. The engineering of MhIHL was initialized by a combinatorial active-site saturation

FIG 1 Enantioselective resolution of Vince lactam by a (�)-�-lactamase for the production of theenantiopure intermediate of abacavir and carbovir.

TABLE 1 Kinetic constants and specific activities for hydrolysis of (�)-�-lactam by (�)-�-lactamases from different microorganisms

Catalysta Km (mM)b kcat (s�1)b Sp act (U/mg) Reference

�lacBJ 0.7 1.8 150 7�lacAP 111 7.8 26.3 8�lacSS 17.8 NR 24.6 15�lacSS-II 53 NR 20.6 15�lacMH NR NR 253.5 6MhIHL 12 200 2,520 31a�lacBJ, (�)-�-lactamase from Bradyrhizobium japonicum; �lacAP, (�)-�-lactamase from Aeropyrum pernix;�lacSS, (�)-�-lactamase from Sulfolobus solfataricus; �lacSS-II, type II (�)-�-lactamase from S. solfataricus;�lacMH, (�)-�-lactamase from M. hydrocarbonoxydans; MhIHL, (�)-�-lactamase in this study. One unit ofenzyme activity was defined as the amount of enzyme that catalyzed the conversion of 1 nM substrate perminute.

bNR, not reported.

Engineering of a (�)-�-Lactamase Applied and Environmental Microbiology

January 2018 Volume 84 Issue 1 e01780-17 aem.asm.org 3

on July 28, 2020 by guesthttp://aem

.asm.org/

Dow

nloaded from

test (20, 34, 35), in which saturation mutagenesis was systematically carried out at allthe relevant sites around the complete binding pocket of the enzyme, not just at oneor two selected sites. It was proven to be an efficient strategy for tuning the catalyticproperties of enantioselectivity and activity (36, 37). The structure and dynamics of abinding pocket could be manipulated at will by reshaping the “lock” in Emil Fischer’slock-and-key hypothesis (or Koshland’s induced fit model) (34, 35). On the basis ofstructural analysis, residues located within 5 Å of the ligand were selected. Amongthese residues, Asp13, Lys78, and Cys111 formed the catalytic triad (38–40); thus, thesethree residues were excluded from mutagenesis. The other residues were chosen as thetarget amino acids for mutation and defined as five potential systematization sites: A(Gln15/Val18), B (His51/Val54), C (Tyr80/Arg81), D (Ala106/107Gln), and E (Phe110/Val112) (Fig. 3). Cooperative mutagenesis of the adjacent residues would more effec-tively affect the local configuration of secondary protein structure than individualresidue substitution (28).

FIG 2 Schematic overview of the protein engineering process in this work.

FIG 3 MhIHL residues chosen for combinatorial active-site saturation test marked in the crystal structure.

Gao et al. Applied and Environmental Microbiology

January 2018 Volume 84 Issue 1 e01780-17 aem.asm.org 4

on July 28, 2020 by guesthttp://aem

.asm.org/

Dow

nloaded from

Five focused libraries at the corresponding sites were created separately andscreened using the colorimetric assay method (16, 41). The method we used here forthe screening of �-lactamases was much more convenient and sensitive than otherscreening methods (42). The assay was used to detect the primary amine group of thehydrolyzed product. If �-lactam was hydrolyzed, the color of the reaction system wouldturn to purple (the color of the control sample was light pink). The whole procedurewas quick and could be accomplished within a short time, thus facilitating the screen-ing process and saving time. The positive mutants after high-throughput screeningwere subsequently analyzed by chiral HPLC to confirm the enhanced enantioselectivity.Six positive mutants, Val54Ser, Val54Leu, Val112Ala, His51Ala, Phe110Ala-Val112Gly,and Val54Thr from libraries B and D, exhibited enantiomeric preference (enantiomericratio [E] � 185) toward (�)-Vince lactam (Table 2). The screening of other libraries failedto yield any mutant with significantly improved enantioselectivity. Kinetic analysis of allsix mutants revealed a trade-off in activity (Table 3).

Compensation of the enantioselectivity/activity trade-off by iterative satura-tion mutagenesis. To rescue the activity loss in the mutants, iterative saturationmutagenesis was applied using the two best variants, Val54Ser and Val54Leu, as thetemplates. The goal was to improve the enzyme activity of the mutants withoutinfluencing the enantioselectivity. To reduce the screening effort, we opted for NDTcodon degeneracy encoding 12 amino acids with diverse side chains (Phe, Leu, Ile, Val,Tyr, His, Asn, Asp, Cys, Arg, Ser, and Gly). All theoretically possible pathways wereexplored. The process was terminated whenever a given library failed to yield variantswith improved activity. After screening the libraries, we unfortunately failed to obtainmutants with improved activity. All the multiple mutants exhibited reduced enzymeactivity compared to Val54Ser and Val54Leu. In conclusion, if both enantioselectivityand activity were considered, the Val54Ser and Val54Leu variants appeared to beacceptable compromises. Although the Val54Ser and Val54Leu demonstrated variantsreduced activity relative to the wild-type enzyme, the Val54Ser variant remained themost active among all of the reported (�)-�-lactamases (Table 1) [the activity of theVal54Ser mutant is 1,280.8 U/mg, and the activity of the most active (�)-�-lactamase,Mhpg, from previous reports is 253.5 U/mg] (12).

TABLE 2 Enantioselectivity of mutant MhIHLa

Protein (�)-Vince lactam (�)-Vince lactam Conversion ratio (%)

Wild type Y Y 100Val54Ser mutant Y N 49.8Val54Leu mutant Y N 48.8Val112Ala mutant Y N 49.2His51Ala mutant Y N 49.1Phe110Ala-Val112Gly mutant Y N 48.3V54Thr mutant Y N 49.7aY, the protein showed activity on (�)-Vince lactam; N, the protein showed no activity on (�)-Vince lactam.The reaction time was long enough to confirm the enantiomeric preference. The ee was �99% for allmutants, and there was no product for the wild type.

TABLE 3 Kinetic characterization and specific activities of MhIHL and its mutants

Protein Km (mM) kcat (s�1)kcat/Km (s�1 ·mM�1)

Sp act(U/mg) E valuea

Wild type 12 � 2 200 � 20 15.77 2,520 � 20 0b

Val54Ser mutant 18 � 1.8 140 � 10 8.2 1,280 � 30 �200Val54Leu mutant 29 � 3 190 � 10 6.7 900 � 20 �200V54Thr mutant 29 � 2 49 � 3 1.7 145 � 9 �200Val112Ala mutant 38 � 5 11 � 0.9 0.3 44 � 3 �200His51Ala mutant 40 � 5 72 � 5 1.8 160 � 10 185Phe110Ala-Val112Gly mutant 39 � 3 7.8 � 1.2 0.2 25 � 3 �200aE value is calculated by E � ln[(1 � Conv.)(1 � ee)]/ln[(1 � Conv.)(1 � ee)], where Conv. is the conversionratio.

bThe reaction time is long enough to calculate the E value.

Engineering of a (�)-�-Lactamase Applied and Environmental Microbiology

January 2018 Volume 84 Issue 1 e01780-17 aem.asm.org 5

on July 28, 2020 by guesthttp://aem

.asm.org/

Dow

nloaded from

Mutants with significant increase in thermostability were generated. The ther-mostability of a protein is another crucial feature for practical applications (43–45). Forthis reason, considerable effort should be made toward the enhancement of thermo-stability by techniques, such as protein engineering. Nevertheless, the difficulty inpredicting the appropriate sites and the optimal amino acid substitution remains amajor challenge. We used two criteria to select the sites for amino acid exchanges:residues with high temperature factors (B-factors), as calculated by B-FITTER, andresidues with high root mean square fluctuation (RMSF) values during the moleculardynamics simulation (46–48). First, we used the crystal structure of MhIHL (PDB code5HA8) to design thermostable variants (31). To analyze the flexibility of the protein, theamino acid B-factor profile of MhIHL was calculated by the B-FITTER software (48). Fromthe B-FITTER output, eight flexible residues with high B-factor values (Glu37, Gly42, Gly55, Val56, Gly57, His68, Glu71, and Ala95) were chosen for saturation mutagenesis.

In addition, we conducted a molecular dynamics simulation to obtain the fluctua-tion information of every individual amino acid during the process. The simulationexperiment provided us with a way to observe the flexibility of the residues from adynamic perspective. After simulation, the RMSF values of all amino acid residues werecalculated. The result indicated three other promising residues (Gly72, Glu141, andArg162) for evolving the thermostability of MhIHL. In total, 11 residues were designedto enhance thermostability. At this time, we noted that most of the mutations residedon surface loops (Fig. 4), where flexibility was expected to be the highest.

Libraries to screen mutants with improved thermostability were constructed suc-cessfully. Thermostability was assessed by measuring the residual activity subsequentto exposure to high temperature (55°C). After screening 35,000 clones, six positiveclones were identified. Sequence analysis confirmed that the mutants were Glu95Phe,Glu95Gln, Glu95Val, Arg162Thr, Glu95Lys, and Gly42Thr-Val56Met-Gly57Val. The param-eter T50

15 (the temperature at which enzyme activity is reduced to 50% after 15 min ofheat treatment) was used to evaluate kinetic stability (49). The activity of the MhIHLvariants was assessed by incubating each enzyme at 30 to 75°C. No significant differ-ences in the residual activity of wild-type MhIHL were observed after heat treatmentbelow 38°C. However, incubation at temperatures above 40°C affected the activitydramatically. The T50

15 of wild-type MhIHL was determined to be 41°C. The T5015 values

of all six positive mutants screened from the libraries were higher than that of the wildtype (Table 4). The best mutant, Glu95Lys, showed an increase in T50

15 by 31°C, whilea minimum change of 7°C was detected for the mutant Gly42Thr-Val56Met-Gly57Val.

Combining mutations evolved for enhanced enantioselectivity and thermosta-bility. After the screening process, we obtained mutants with improved enantioselec-tivity and mutants with enhanced thermostability separately. Since beneficial muta-tions often demonstrate an additive effect, combining mutations appeared to be a

FIG 4 Secondary-structure topology of MhIHL. The nomenclature of the strands is taken from thecanonical �/�-hydrolase fold. All the positions that were chosen to enhance the thermostability arerepresented as red circles, the amino acids of the catalytic triad are represented as black circles, and thetermini of the protein are represented as C and N.

Gao et al. Applied and Environmental Microbiology

January 2018 Volume 84 Issue 1 e01780-17 aem.asm.org 6

on July 28, 2020 by guesthttp://aem

.asm.org/

Dow

nloaded from

promising option for improving the overall performance of the enzyme (50). Therefore,the Glu95Gln, Glu95Val, Arg162Thr, and Glu95Lys mutants were chosen as the tem-plates to conduct the site-directed mutagenesis experiment. The mutants Val54Ser andVal54Leu were incorporated into the mutant templates to generate eight doublemutants for further study. The results of the combinatorial mutagenesis are summa-rized in Table 5. Notably, all the mutations exhibited better enantioselectivity (E � 178)and increased thermostability (increase in T50

15 by 20 to 29°C) relative to the wild-typeprotein. This was a predictable phenomenon, because the residues involved inthe enantioselectivity are located around the active-site pocket, but those relatedto the thermal robustness of protein are located on the protein surface, distant fromthe active-site pocket.

The best properties were observed from the Glu95Lys-Val54Ser and Arg162Thr-Val54Leu mutants. The optimal temperatures for catalytic activity of Glu95Lys-Val54Serand Arg162Thr-Val54Leu were 35°C and 40°C, respectively (Fig. 5). Compared to thewild-type protein, they all showed an increased optimal temperature (10 and 15°Chigher, respectively). On the basis of enantioselectivity, thermostability, and activity, itcan be concluded that the Glu95Lys-Val54Ser mutant may be suitable for preparationof the optically pure intermediate (�)-Vince lactam.

DISCUSSION

(�)-Vince lactam is a key intermediate in the synthesis of abacavir and carbovir,which are powerful selective DNA polymerase inhibitors used to treat HIV and hepatitisB virus (1). An advanced method for obtaining (�)-Vince lactam would be the directhydrolysis of racemic Vince lactam by catalysts with high efficiency and good enanti-oselectivity in an environmentally friendly way. (�)-�-Lactamase is a small enzymesubclassification in amidase, which specifically catalyzes the hydrolysis of (�)-Vincelactam. The discovery and identification of (�)-�-lactamases have made it a much moreattractive approach to obtain (�)-Vince lactam enzymatically. In our previous work, a(�)-�-lactamase from M. hydrocarbonoxydans was identified (16). Based on the bioin-formatics analysis, the enzyme was assigned to one of the isochorismatase-like super-family members, indicating its promiscuity. This new type enzyme shows the highest

TABLE 4 T5015 values of MhIHL and its mutants

Protein T5015 (°C)a

Wild type 41Glu95Phe mutant 60Glu95Gln mutant 70Glu95Val mutant 58Arg162Thr mutant 57Glu95Lys mutant 72Gly42Thr-Val56Met-Gly57Val mutant 48aT50

15, the temperature at which 50% of the enzyme activity is lost following heat treatment for 15 min. Theenzyme was incubated at different temperatures (30 to 75°C) for 15 min, followed by measuring of theresidual activity.

TABLE 5 Kinetic characterization and thermostability determination of best MhIHLvariants

Protein Km (mM) kcat (s�1)kcat/Km (s�1 ·mM�1) T50

15 (°C) E value

Wild type 12 � 2 200 � 20 15.77 41 0Glu95Gln-Val54Ser mutant 24 � 2 143 � 10 5.9 67 178Glu95Gln-Val54Leu mutant 28.1 � 1.2 101 � 7 3.6 65 199Glu95Val-Val54Ser mutant 18 � 2 120 � 13 6.5 62 187Glu95Val-Val54Leu mutant 26.3 � 1.1 168 � 8 6.4 61 �200Arg162Thr-Val54Ser mutant 18 � 2 130 � 10 7.2 66 �200Arg162Thr-Val54Leu mutant 20.2 � 0.9 159 � 10 7.9 69 �200Glu95Lys-Val54Ser mutant 20 � 2 164 � 10 8.2 70 �200Glu95Lys-Val54Leu mutant 20.2 � 1.9 123 � 8 6.1 68 �200

Engineering of a (�)-�-Lactamase Applied and Environmental Microbiology

January 2018 Volume 84 Issue 1 e01780-17 aem.asm.org 7

on July 28, 2020 by guesthttp://aem

.asm.org/

Dow

nloaded from

(�)-�-lactamase activity among all the reported (�)-�-lactamases, making it an out-standing candidate for obtaining optically pure (�)-Vince lactam (Table 1). However,the enzyme also displays (�)-�-lactamase activity when the (�)-enantiomer runs out(31), i.e., the enzyme exhibits poor enantioselectivity. In addition, the enzyme was notquite thermostable. The enzyme activity decreases dramatically when the temperatureis higher than 35°C. Considering the high enzyme activity, it is worthwhile to engineerthe protein from the application point of view. To better understand the catalyticmechanism and facilitate the protein engineering process, we determined the crystalstructures of the apo, (�)-�-lactam-bound, and (�)-�-lactam-bound forms of theenzyme (PDB codes 5HA8, 5HWH, and 5HWG, respectively). Structural insights into the�-lactamase activity and substrate enantioselectivity of the enzyme were well studied(31). In this paper, both the enantioselectivity and thermostability were tackled byusing a semirational design based on knowledge about the structure and mechanism.

Two promising mutants, Val54Leu and Val54Ser, were screened out after performingthe combinatorial active-site saturation test strategy on the protein. Both mutantsshowed excellent enantioselectivity but at the cost of activity loss (Val54Leu andVal54Ser retain one-third and one-half of the original activity, respectively). To under-stand the reason behind the enantioselectivity/activity trade-off, the kinetics of themutants was analyzed (Table 3). For the Val54Ser mutant, the decrease in the catalyticefficiency is caused by both lower affinity and a lower rate constant, but the activity lossof the Val54Leu mutant is mainly attributed to lower affinity. In our previous study, wementioned that when the structure of MhIHL was superimposed with other homologsfrom the isochorismatase-like superfamily, the dramatic feature we observed was thatour enzyme lacked a loop which existed in other homologs serving as an active site“cover” (31) (shown in Fig. 6). We suggested that this missing loop motif was correlatedwith the loading of the substrate or the release of the product. As we proposed (Fig. 7),the residue Val 54 is right on the way to the binding pocket, and the substrate loadingpath could be influenced by the mutations in Val54. When the valine is changed toleucine, which is one carbon longer, the side chain of Val54Leu may form a sterichindrance which leads to the activity decrease for both enantiomers. Since the bindingof the (�)-enantiomer is already not favorable (31), the addition of the steric hindrancecould result in a loss of activity by a great extent. The steric hindrance and unfavorablebinding for the (�)-enantiomer result in much more activity loss than the activity lossfor the other enantiomer, thus contributing to the better enantioselectivity of theVal54Leu mutant. When the valine is changed to serine, the property around the

FIG 5 Temperature dependence of wild-type and mutant MhIHL. Black squares, wild type; red circles,Glu95Lys-Val54Ser mutant; blue triangles, Arg162Thr-Val54Leu mutant.

Gao et al. Applied and Environmental Microbiology

January 2018 Volume 84 Issue 1 e01780-17 aem.asm.org 8

on July 28, 2020 by guesthttp://aem

.asm.org/

Dow

nloaded from

binding pocket is changed by substitution of the hydrophilic residue. The polar sidechain of the mutant residue may have interactions with the polar part of the substrate,which hinders the substrate from getting into the right position for catalysis. Theimpact is more significant when the valine is changed to threonine, which has onemore carbon than serine (the activity is reduced to about 10%). In this situation, exceptfor the interaction between the polar side chain with the polar part of the substrate,there could be some degree of steric hindrance as well.

We tried to rescue the activity loss caused by the trade-off with enantioselectivity.Iterative saturation mutagenesis was applied to the two best variants, Val54Ser andVal54Leu. Unfortunately, we have not achieved any mutants with higher activity.Compensation of the enantioselectivity/activity trade-off in the protein engineering isan important work to achieve better mutants (51). Yu et al. succeeded in rescuing the

FIG 6 Comparison of the active pockets between MhIHL (green, PDB 5HWH) and the representative ofIHL family proteins: nicotinamidase-nicotinamide complex (magenta, PDB 3O94). The regions corre-sponding to the active-site “covers” are indicated. The substrate (�)-Vince lactam is shown in sticks(nitrogen, oxygen, carbon, and hydrogen are colored blue, red, green, and white, respectively), and Val54is shown in yellow sticks.

FIG 7 Proposed substrate loading path to the binding pocket. The residues Asp13, Gln107, Ala111(Cys111 in wild type), and Val54 are shown in sticks (nitrogen, oxygen, carbon, and hydrogen are coloredblue, red, cyan, and white, respectively). The yellow arrow indicates the direction how the substrateenters the active site.

Engineering of a (�)-�-Lactamase Applied and Environmental Microbiology

January 2018 Volume 84 Issue 1 e01780-17 aem.asm.org 9

on July 28, 2020 by guesthttp://aem

.asm.org/

Dow

nloaded from

activity loss in directed evolution by using the mutants with higher activity as thetemplate to undergo site-directed saturation mutagenesis at specific hot spots toscreen for better enantioselectivity (52). More useful strategies, such as computationaldesign and error-prone PCR, could be tried to save the activity loss of our mutantenzyme in the future study (51).

In the thermostability study, target residues were chosen based on our calculationsfrom both a static (B-FITTER based on the crystal structure) and a dynamic (RMSF valueswere calculated based on molecular dynamics [MD] simulations) way. The RMSF resultsfrom molecular dynamics simulation represent the flexibility of every individual residuefrom a mobile perspective rather than a static perspective in the crystal lattice. Weobtained six mutants which showed higher T50

15 than the wild type. The best mutant,Glu95Lys, exhibited a 31°C increase in T50

15. A simple analysis regarding the sources ofenhanced thermostability is offered here. In the best mutant, Glu95Lys, the residue waschanged from a negatively charged Glu to a positively charged Lys. We suggested thatthis negative-to-positive change may contribute to the redistribution of the surfacecharge on the protein (53). Thus, it could help stabilize loop structures through a varietyof interactions, such as salt bridges. Recent experimental and theoretical work sug-gested that large stability enhancements might be obtained through the design ofsurface-charge distribution (54). A few genetic algorithms were proposed and em-ployed to select small sets of interacting sites to be used in an in vitro-directedevolution process that addressed the achievement of protein variants with enhancedstability (55). In addition, the rigidity of the residues affected protein kinetic stabilitysignificantly (47, 56, 57). It was wise to target the flexible residues or segment on theprotein surface for improved stability. However, sometimes enhancing the rigidity ofthe flexible segment within the active site may also work well (47).

Then, the separately evolved mutants were combined to generate new ones withthe best overall performance. As we predicted, the combined double mutants main-tained both good enantioselectivity and thermostability. Our work once again showsthe efficiency of the combinatorial active-site saturation test strategy in engineeringenantioselectivity. Another phenomenon we noticed in the engineering process is thatcharged flexible residues could be prominent targets to enhance the thermal robust-ness of the protein, as the distribution of surface charge may be related to undesirableprotein aggregation or precipitation. In conclusion, our protein engineering work hasgenerated mutants with good catalytic profiles considering enantioselectivity, thermo-stability, and activity, and it provides a sound example for the engineering of (�)-�-lactamases. Optimization of several enzyme properties by protein engineering tech-nology remains a great challenge (19, 25). Whether through sequential optimization,e.g., directed evolution of one property followed by another property, or evolution ofthe three properties simultaneously, multiproperty optimization of a protein still has along way to go. More novel and innovative strategies are needed to facilitate theprocess of protein engineering.

MATERIALS AND METHODSMaterials. Vince lactam was purchased from Sigma-Aldrich (Munich, Germany). Isopropanol and

n-hexane of high-performance liquid chromatography (HPLC) grade were purchased from Thermo FisherScientific (Waltham, MA, USA). Other reagents were purchased from Acros (Beijing, China). The gene ofMhIHL (GenBank accession number KT335970) from M. hydrocarbonoxydans (conserved in GeneralMicrobiological Culture Collection Center [CGMCC], strain collection number 6.3423) was cloned into thevector pET28a(�) (Novagen, Darmstadt, Germany), with a 6�His tag at the N terminus, in our previousstudy (16).

MD simulation and B-FITTER. The crystal structure of wild-type MhIHL (PDB code 5HA8) wasobtained in our previous work (31). To explore the residues related to the thermostability of the wild-typeprotein, a 30-ns molecular dynamics (MD) simulation was performed with the GROMACS softwarepackage, version 5.0.7 (58). The initial structure was solvated with transferable intermolecularpotential with 3 points (TIP3P) water molecules in a box. A sufficient number of Na ions were addedto neutralize the negative charges in the system. Periodic boundary conditions were applied in alldirections. A steepest-descent algorithm and conjugate gradient algorithm were performed insuccession to minimize the energy of the system and relax the water molecules. MD simulation wasperformed on the whole system for 30 ns at 300 K. All bond lengths were constrained using the

Gao et al. Applied and Environmental Microbiology

January 2018 Volume 84 Issue 1 e01780-17 aem.asm.org 10

on July 28, 2020 by guesthttp://aem

.asm.org/

Dow

nloaded from

LINear Constraint Solver (LINCS) algorithm. The cutoff value for van der Waals interactions was setat 1 nm, and electrostatic interactions were calculated using a particle mesh Ewald algorithm. Thetime step of the simulations was set at 2 fs. Postprocessing and analysis were performed usingstandard GROMACS tools. To investigate the stability of the protein, the root mean square fluctu-ation (RMSF) values were calculated.

B-FITTER is a user-friendly computer aid when applying the B-FIT method in the quest to enhancethe thermal robustness of proteins, which can be related to thermodynamic stability. This programcalculates the amino acid B-factor as an average of the B-factors of all of the atoms of an amino acidin a given protein, excluding hydrogen. It is able to recognize the existence of nonresolved atomsand takes them into account for the calculation of the average amino acid B factors (20). The inputwas a .pdb file, and the output was the ranking of the 20 amino acid residues with the highestB-values.

Primer design and library creation of MhIHL. Primer design and library construction dependedupon the experiments specifically performed. For the combinatorial active-site saturation test experi-ment, the residues involved were divided into five groups. For the experiment designed to enhancethermostability, the residues involved were divided into 8 groups. For the experiments designed tocombine mutants that were evolved for enhanced thermostability and mutants with improved enanti-oselectivity, primers were designed specifically. The plasmid pET28-MhIHL in our previous study wasused as the first template (16). The mutants were generated by the QuikChange site-directed mutagen-esis method using specific primers or QuikChange PCR method using degenerate codons (59). The PCRtemplate was digested by DpnI, and E. coli Rosetta(DE3) was transformed with the product to create thelibrary for screening.

Screening procedures. Colonies were picked up and inoculated into 96-deep-well plates containing200 �l of lysogeny broth (LB) medium and kanamycin (50 �g/ml). Oversampling was performed toensure �95% coverage of the library (for the study containing one mutant position, more than 100colonies were screened; for the libraries containing two mutant positions, more than 4,000 colonies werescreened). The inoculated media were cultured overnight at 37°C with shaking. Then, 1 mM isopropyl-�-D-thiogalactopyranoside (IPTG) was added directly to the culture for protein expression for 10 h at 25°Cwith shaking. The cell pellets were harvested by centrifugation for 15 min at 2,000 rpm. Then, the cellswere suspended in 200 �l of water. The 96-deep-well plates were incubated at 55°C (after heating at 55°Cfor 15 min, the wild-type MhIHL retained about 10% activity; therefore, we selected this condition for thehigh-throughput screening of thermostability) for 15 min and cooled at 4°C for 20 min, followed byequilibration at room temperature for 15 min. The heat-treated cells were centrifuged to remove thewater. Then, 200 �l of 50 mM Vince lactam was added into the wells, and the reaction was performedat 30°C for 5 min. Then, the reaction suspension (80 �l) was used for a colorimetric assay by using thehigh-throughput screening method (16, 41). The suspensions after the reaction from the 96-well plateswere mixed with 10 �l of saturated sodium bicarbonate, 32 �l of acetone, and 40 �l of sodiumnitroprusside (60 mM) sequentially. After mixing, the solution was placed at room temperature for 10 minfor observation by eye. The original color of the reaction was light red. If Vince lactam was hydrolyzedinto 4-aminocyclopent-2-ene-1-carboxylic acid, a substance with a primary amine group, the color of thereaction system would change to purple. The degree of the color change reflected the residual activityof the �-lactamase. The positive mutants were further identified by chiral high-performance liquidchromatography (HPLC).

Libraries designed to enhance enantioselectivity were screened using the same procedure asmentioned above except that there was no heat treatment of the cells.

Protein expression and purification. The wild-type and positive mutants were inoculated in 5 mlof LB medium containing kanamycin (50 �g/ml), cultured overnight at 37°C with shaking, and thenscaled up to 500 ml. IPTG (1.0 mM) was added to induce the protein expression when the optical densityat 600 nm (OD600) value of the medium reached 0.8 to 1.0. The cell cultures continued to grow for 10h at 25°C before being harvested by centrifugation. The cell pellets were disrupted by sonication on ice,and then the cell debris was removed by centrifugation. The proteins were purified by immobilized metalaffinity chromatography using a 6�His tag (16). The eluted protein sample was concentrated byultrafiltration using YM10 membranes (Amicon) and then loaded onto a Superdex 200 column (GEHealthcare) and eluted with 50 mM Tris (pH 7.8), 500 mM NaCl, and 5 mM dithiothreitol (DTT). Fractionscontaining pure protein were collected. The protein concentrations were determined by using a ThermoNanoDrop 2000 at 280 nm. The concentration values obtained from the NanoDrop were calibrated usingthe calculated extinction coefficient (22,460 M�1 · cm�1).

Enzyme activity assays. The activity of (�)-�-lactamase was assayed by adding 5 �g of pure enzymeto 1,000 �l of the 1.0 M substrate solution (Tris-HCl buffer [pH 7.5]). The reaction mixture was incubatedat 30°C for 1 min and was then terminated by extraction with 1,000 of �l ethyl acetate. The decrease ofthe substrate was detected by HPLC. The extraction solution (10 �l) was subjected to a chiral AD-Hcolumn (25 cm by 4.6 mm; Daicel, Inc.) and eluted with a mobile phase consisting of 93% n-hexane and7% isopropanol (volume ratio) at a flow rate of 0.75 ml/min. The UV absorbance of the eluted �-lactamwas at 230 nm. One unit of (�)-�-lactamase is defined as the amount of enzyme required to hydrolyze1 nmol substrate per minute.

Enzyme stability determination. Heat treatment of purified protein was carried out by incubatingthe protein in a 0.2-ml PCR tube in a programmable thermal cycler for precise temperature control.Proteins (50 �l of 0.1 mg/ml in 50 mM Tris-HCl [pH 7.8]) were heated at different temperatures (30 to75°C) for 15 min, cooled at 4°C for 15 min, and equilibrated at room temperature for 15 min. Sampleswere centrifuged to remove any aggregated protein before the enzymatic activity assay. The activity at

Engineering of a (�)-�-Lactamase Applied and Environmental Microbiology

January 2018 Volume 84 Issue 1 e01780-17 aem.asm.org 11

on July 28, 2020 by guesthttp://aem

.asm.org/

Dow

nloaded from

30°C without heat treatment was considered to be 100%. The T5015 value is the temperature at which

enzyme activity is reduced to 50% after 15 min of heat treatment. The T5015 values of the wild-type

protein and mutant proteins were determined by the above-described procedure.Measurement of kinetic parameters of purified enzyme. In the kinetic studies, all the conversion

ratios were controlled within 10% to make the determinations accurate. The initial velocity region (linearregion) of the enzymatic reaction was determined prior to conducting real kinetic studies, and 1 min waschosen as the reaction time. Pure mutant enzymes were added to Tris-HCl buffer (50 mM [pH 7.8]), witha total volume of 1,000 �l containing Vince lactam at various concentrations (2 to 200 mM). The reactionwas performed at 30°C for 1 min with shaking (1,200 rpm) and terminated by extraction with 1,000 �lof ethyl acetate. The extraction solution (10 �l) was analyzed by HPLC. Initial velocities were calculatedby dividing the consumption of the substrate by the reaction time. The kinetic parameters were obtainedafter the curve fitting based on Michaelis-Menten equation (60, 61).

ACKNOWLEDGMENTSThis work was supported by the Fundamental Research Funds for the Central

Universities of China (grant YS1407).We express our gratitude to Klinman lab group members (Judith Klinman, Ian Barr,

Shenshen Hu, Eric Koehn, Adam Offenbacher, Emily Thompson, Wen Zhu, and Ana M.Martins) from the Department of Chemistry and California Institute for QuantitativeBiosciences, University of California, Berkeley, for helping analyze and explain theunderlying mechanism of mutants achieved in the paper.

REFERENCES1. Boutureira O, Matheu MI, Diaz Y, Castillon S. 2013. Advances in the

enantioselective synthesis of carbocyclic nucleosides. Chem Soc Rev42:5056 –5072. https://doi.org/10.1039/c3cs00003f.

2. Holt-Tiffin KE. 2009. (�)- and (�)-2-azabicyclo[2.2.1]hept-5-en-3-one. Ex-tremely useful synthons. Chim Oggi 27:23–25.

3. Marquez VE, Lim MI. 1986. Carbocyclic nucleosides. Med Res Rev 6:1– 40.https://doi.org/10.1002/med.2610060102.

4. Singh R, Vince R. 2012. 2-Azabicyclo[2.2.1]hept-5-en-3-one: chemicalprofile of a versatile synthetic building block and its impact on thedevelopment of therapeutics. Chem Rev 112:4642– 4686. https://doi.org/10.1021/cr2004822.

5. Taylor SJC, Sutherland AG, Lee CWR, Thomas S, Roberts SM, Evans C.1990. Chemoenzymatic synthesis of (�)-carbovir utilizing a whole cellcatalyzed resolution of 2-azabicyclo[2.2.1]hept-5-en-3-one. J Chem SocChem Commun 16:1120 –1121. https://doi.org/10.1039/C39900001120.

6. Toogood HS, Brown RC, Line K, Keene PA, Taylor SJC, McCague R,Littlechild JA. 2004. The use of a thermostable signature amidase in theresolution of the bicyclic synthon (rac)-gamma-lactam. Tetrahedron Lett60:711–716. https://doi.org/10.1016/j.tet.2003.11.064.

7. Gao S, Zhu S, Huang R, Lu Y, Zheng G. 2015. Efficient synthesis of theintermediate of abacavir and carbovir using a novel (�)-gamma-lactamase as a catalyst. Bioorg Med Chem Lett 25:3878 –3881. https://doi.org/10.1016/j.bmcl.2015.07.054.

8. Ren L, Zhu S, Shi Y, Gao S, Zheng G. 2015. Enantioselective resolution ofgamma-lactam by a novel thermostable type II (�)-gamma-lactamasefrom the hyperthermophilic archaeon Aeropyrum pernix. Appl BiochemBiotechnol 176:170 –184. https://doi.org/10.1007/s12010-015-1565-7.

9. Taylor SJ, Brown RC, Keene PA, Taylor IN. 1999. Novel screeningmethods–the key to cloning commercially successful biocatalysts. BioorgMed Chem 7:2163–2168. https://doi.org/10.1016/S0968-0896(99)00146-7.

10. Taylor SJC, Mccague R, Wisdom R, Lee C, Dickson K, Ruecroft G, ObrienF, Littlechild J, Bevan J, Roberts SM, Evans CT. 1993. Development of thebiocatalytic resolution of 2-azabicyclo[2.2.1]hept-5-en-3-one as an entryto single-enantiomer carbocyclic nucleosides. Tetrahedron: Asymmetry4:1117–1128. https://doi.org/10.1016/S0957-4166(00)80218-9.

11. Wang J, Zhu J, Wu S. 2015. Immobilization on macroporous resin makesE. coli RutB a robust catalyst for production of (�) Vince lactam. ApplMicrobiol Biotechnol 99:4691– 4700. https://doi.org/10.1007/s00253-014-6247-9.

12. Wang J, Zhu Y, Zhao G, Zhu J, Wu S. 2015. Characterization of arecombinant (�)-gamma-lactamase from Microbacterium hydrocarbon-oxydans which provides evidence that two enantiocomplementarygamma-lactamases are in the strain. Appl Microbiol Biotechnol 99:3069 –3080. https://doi.org/10.1007/s00253-014-6114-8.

13. Xue TY, Xu GC, Han RZ, Ni Y. 2015. Soluble expression of (�)-gamma-lactamase in Bacillus subtilis for the enantioselective preparation of

abacavir precursor. Appl Biochem Biotechnol 176:1687–1699. https://doi.org/10.1007/s12010-015-1670-7.

14. Zhu S, Gong C, Song D, Gao S, Zheng G. 2012. Discovery of a novel(�)-gamma-lactamase from Bradyrhizobium japonicum USDA 6 by ratio-nal genome mining. Appl Environ Microbiol 78:7492–7495. https://doi.org/10.1128/AEM.01398-12.

15. Zhu S, Huang R, Gao S, Li X, Zheng G. 2016. Discovery and character-ization of a second extremely thermostable (�)-gamma-lactamase fromSulfolobus solfataricus P2. J Biosci Bioeng 121:484 – 490. https://doi.org/10.1016/j.jbiosc.2015.09.012.

16. Gao S, Huang R, Zhu S, Li H, Zheng G. 2016. Identification and charac-terization of a novel (�)-gamma-lactamase from Microbacterium hydro-carbonoxydans. Appl Microbiol Biotechnol 100:9543–9553. https://doi.org/10.1007/s00253-016-7643-0.

17. Quin MB, Schmidt-Dannert C. 2011. Engineering of biocatalysts–fromevolution to creation. ACS Catal 1:1017–1021. https://doi.org/10.1021/cs200217t.

18. Behrens GA, Hummel A, Padhi SK, Schätzle S, Bornscheuer UT. 2011. Dis-covery and protein engineering of biocatalysts for organic synthesis. AdvSynth Catal 353:2191–2215. https://doi.org/10.1002/adsc.201100446.

19. Bommarius AS, Blum JK, Abrahamson MJ. 2011. Status of protein engi-neering for biocatalysts: how to design an industrially useful biocatalyst.Adv Synth Catal 15:194 –200. https://doi.org/10.1016/j.cbpa.2010.11.011.

20. Reetz MT. 2011. Laboratory evolution of stereoselective enzymes: aprolific source of catalysts for asymmetric reactions. Angew Chem Int EdEngl 50:138 –174. https://doi.org/10.1002/anie.201000826.

21. Reetz MT. 2013. Biocatalysis in organic chemistry and biotechnology:past, present, and future. J Am Chem Soc 135:12480 –12496. https://doi.org/10.1021/ja405051f.

22. Reetz MT, Kahakeaw D, Sanchis J. 2009. Shedding light on the efficacy oflaboratory evolution based on iterative saturation mutagenesis. MolBiosyst 5:115–122. https://doi.org/10.1039/B814862G.

23. Sletten EM, Bertozzi CR. 2009. Bioorthogonal chemistry: fishing for se-lectivity in a sea of functionality. Angew Chem Int Ed Engl 48:6974 – 6998. https://doi.org/10.1002/anie.200900942.

24. Cao LC, Chen R, Xie W, Liu YH. 2015. Enhancing the thermostability offeruloyl esterase EstF27 by directed evolution and the underlying struc-tural basis. J Agric Food Chem 63:8225– 8233. https://doi.org/10.1021/acs.jafc.5b03424.

25. Li G, Zhang H, Sun Z, Liu X, Reetz MT. 2016. Multiparameter optimizationin directed evolution: engineering thermostability, enantioselectivity,and activity of an epoxide hydrolase. ACS Catal 6:3679 –3687. https://doi.org/10.1021/acscatal.6b01113.

26. Reetz MT, Bocola M, Wang LW, Sanchis J, Cronin A, Arand M, Zou J,Archelas A, Bottalla AL, Naworyta A, Mowbray SL. 2009. Directed evolu-tion of an enantioselective epoxide hydrolase: uncovering the source of

Gao et al. Applied and Environmental Microbiology

January 2018 Volume 84 Issue 1 e01780-17 aem.asm.org 12

on July 28, 2020 by guesthttp://aem

.asm.org/

Dow

nloaded from

enantioselectivity at each evolutionary stage. J Am Chem Soc 131:7334 –7343. https://doi.org/10.1021/ja809673d.

27. Ruff AJ, Dennig A, Wirtz G, Blanusa M, Schwaneberg U. 2012. Flowcytometer-based high-throughput screening system for accelerated di-rected evolution of P450 monooxygenases. ACS Catal 2:2724 –2728.https://doi.org/10.1021/cs300115d.

28. Zhang D, Chen X, Chi J, Feng J, Wu Q, Zhu D. 2015. Semi-rationalengineering a carbonyl reductase for the enantioselective reductionof �-amino ketones. ACS Catal 5:2452–2457. https://doi.org/10.1021/acscatal.5b00226.

29. Guo F, Franzen S, Ye L, Gu J, Yu H. 2014. Controlling enantioselectivity ofesterase in asymmetric hydrolysis of aryl prochiral diesters by introduc-ing aromatic interactions. Biotechnol Bioeng 111:1729 –1739. https://doi.org/10.1002/bit.25249.

30. Li A, Ye L, Yang X, Wang B, Yang C, Yu H. 2016. Reconstruction of thecatalytic pocket and enzyme-substrate interactions to enhance the cat-alytic efficiency of a short-chain dehydrogenase/reductase. Chem-CatChem 8:3229 –3233. https://doi.org/10.1002/cctc.201600921.

31. Gao S, Zhou Y, Zhang W, Wang W, Yu Y, Mu Y, Wang H, Gong X, ZhengG, Feng Y. 2017. Structural insights into the gamma-lactamase activityand substrate enantioselectivity of an isochorismatase-like hydrolasefrom Microbacterium hydrocarbonoxydans. Sci Rep 7:44542. https://doi.org/10.1038/srep44542.

32. Marchler-Bauer A, Derbyshire MK, Gonzales NR, Lu S, Chitsaz F, Geer LY,Geer RC, He J, Gwadz M, Hurwitz DI, Lanczycki CJ, Lu F, Marchler GH,Song JS, Thanki N, Wang Z, Yamashita RA, Zhang D, Zheng C, Bryant SH.2015. CDD: NCBI’s Conserved Domain Database. Nucleic Acids Res 43:222–226. https://doi.org/10.1093/nar/gku1221.

33. Haki GD, Rakshit SK. 2003. Developments in industrially important ther-mostable enzymes: a review. Bioresour Technol 89:17–34. https://doi.org/10.1016/S0960-8524(03)00033-6.

34. Reetz MT, Wang LW, Bocola M. 2006. Directed evolution of enantiose-lective enzymes: iterative cycles of CASTing for probing protein-sequence space. Angew Chem Int Ed Engl 45:1236 –1241. https://doi.org/10.1002/anie.200502746.

35. Reetz MT, Bocola M, Carballeira JD, Zha D, Vogel A. 2005. Expanding therange of substrate acceptance of enzymes: combinatorial active-sitesaturation test. Angew Chem Int Ed Engl 44:4192– 4196. https://doi.org/10.1002/anie.200500767.

36. Reetz MT, Wilensek S, Zha D, Jaeger KE. 2001. Directed evolution of anenantioselective enzyme through combinatorial multiple-cassette mu-tagenesis. Angew Chem Int Ed Engl 40:3589 –3591. https://doi.org/10.1002/1521-3773(20011001)40:19�3589::AID-ANIE3589�3.0.CO;2-X.

37. Liebeton K, Zonta A, Schimossek K, Nardini M, Lang D, Dijkstra BW, ReetzMT, Jaeger KE. 2000. Directed evolution of an enantioselective lipase.Chem Biol 7:709 –718. https://doi.org/10.1016/S1074-5521(00)00015-6.

38. Goral AM, Tkaczuk KL, Chruszcz M, Kagan O, Savchenko A, Minor W.2012. Crystal structure of a putative isochorismatase hydrolase fromOleispira antarctica. J Struct Funct Genomics 13:27–36. https://doi.org/10.1007/s10969-012-9127-5.

39. Parsons JF, Calabrese K, Eisenstein E, Ladner JE. 2003. Structure andmechanism of Pseudomonas aeruginosa PhzD, an isochorismatase fromthe phenazine biosynthetic pathway. Biochemistry 42:5684 –5693.https://doi.org/10.1021/bi027385d.

40. Romão MJ, Turk D, Gomis-Ruth FX, Huber R, Schumacher G, Mollering H,Russmann L. 1992. Crystal structure analysis, refinement and enzymaticreaction mechanism of N-carbamoylsarcosine amidohydrolase from Ar-throbacter sp. at 2.0 Å resolution. J Mol Biol 226:1111–1130. https://doi.org/10.1016/0022-2836(92)91056-U.

41. Stoyanovsky DA, Clancy R, Cederbaum AI. 1999. Decomposition of so-dium trioxodinitrate (Angeli’s salt) to hydroxyl radical. J Am Chem Soc121:5093–5094. https://doi.org/10.1021/ja990318v.

42. He YC, Ma CL, Xu JH, Zhou L. 2011. A high-throughput screeningstrategy for nitrile-hydrolyzing enzymes based on ferric hydroxamatespectrophotometry. Appl Microbiol Biotechnol 89:817– 823. https://doi.org/10.1007/s00253-010-2977-5.

43. Eijsink VG, Gaseidnes S, Borchert TV, van den Burg B. 2005. Directed

evolution of enzyme stability. Biomol Eng 22:21–30. https://doi.org/10.1016/j.bioeng.2004.12.003.

44. Hoseki J, Okamoto A, Takada N, Suenaga A, Futatsugi N, Konagaya A,Taiji M, Yano T, Kuramitsu S, Kagamiyama H. 2003. Increased rigidity ofdomain structures enhances the stability of a mutant enzyme created bydirected evolution. Biochemistry 42:14469 –14475. https://doi.org/10.1021/bi034776z.

45. Reetz MT, Carballeira JD, Vogel A. 2006. Iterative saturation mutagenesison the basis of B factors as a strategy for increasing protein thermosta-bility. Angew Chem Int Ed Engl 45:7745–7751. https://doi.org/10.1002/anie.200602795.

46. Yu H, Yan Y, Zhang C, Dalby PA. 2017. Two strategies to engineer flexibleloops for improved enzyme thermostability. Sci Rep 7:41212. https://doi.org/10.1038/srep41212.

47. Xie Y, An J, Yang G, Wu G, Zhang Y, Cui L, Feng Y. 2014. Enhancedenzyme kinetic stability by increasing rigidity within the active site. J BiolChem 289:7994 – 8006. https://doi.org/10.1074/jbc.M113.536045.

48. Reetz MT, Carballeira JD. 2007. Iterative saturation mutagenesis (ISM) forrapid directed evolution of functional enzymes. Nat Protoc 2:891–903.https://doi.org/10.1038/nprot.2007.72.

49. Stephens DE, Rumbold K, Permaul K, Prior BA, Singh S. 2007. Directedevolution of the thermostable xylanase from Thermomyces lanugino-sus. J Biotechnol 127:348 –354. https://doi.org/10.1016/j.jbiotec.2006.06.015.

50. Fujii R, Nakagawa Y, Hiratake J, Sogabe A, Sakata K. 2005. Directedevolution of Pseudomonas aeruginosa lipase for improved amide-hydrolyzing activity. Protein Eng Des Sel 18:93–101. https://doi.org/10.1093/protein/gzi001.

51. Dalby PA. 2011. Strategy and success for the directed evolution ofenzymes. Curr Opin Struct Biol 21:473– 480. https://doi.org/10.1016/j.sbi.2011.05.003.

52. Guo F, Xu H, Xu H, Yu H. 2013. Compensation of theenantioselectivity-activity trade-off in the directed evolution of anesterase from Rhodobacter sphaeroides by site-directed saturationmutagenesis. Appl Microbiol Biotechnol 97:3355–3362. https://doi.org/10.1007/s00253-012-4516-z.

53. Strickler SS, Gribenko AV, Gribenko AV, Keiffer TR, Tomlinson J, Reihle T,Loladze VV, Makhatadze GI. 2006. Protein stability and surface electrostatics:a charged relationship. Biochemistry 45:2761–2766. https://doi.org/10.1021/bi0600143.

54. Lee CW, Wang HJ, Hwang JK, Tseng CP. 2014. Protein thermal stabilityenhancement by designing salt bridges: a combined computational andexperimental study. PLoS One 9:e112751. https://doi.org/10.1371/journal.pone.0112751.

55. Ibarra-Molero B, Sanchez-Ruiz JM. 2002. Genetic algorithm to designstabilizing surface-charge distributions in proteins. J Phys Chem B 106:6609 – 6613. https://doi.org/10.1021/jp020483o.

56. Rader AJ. 2009. Thermostability in rubredoxin and its relationship tomechanical rigidity. Phys Biol 7:16002. https://doi.org/10.1088/1478-3975/7/1/016002.

57. Wray JW, Baase WA, Lindstrom JD, Weaver LH, Poteete AR, Matthews BW.1999. Structural analysis of a non-contiguous second-site revertant in T4lysozyme shows that increasing the rigidity of a protein can enhance itsstability. J Mol Biol 292:1111–1120. https://doi.org/10.1006/jmbi.1999.3102.

58. Van Der Spoel D, Lindahl E, Hess B, Groenhof G, Mark AE, Berendsen HJ.2005. GROMACS: fast, flexible, and free. J Comput Chem 26:1701–1718.https://doi.org/10.1002/jcc.20291.

59. Hogrefe HH, Cline J, Youngblood GL, Allen RM. 2002. Creating random-ized amino acid libraries with the QuikChange multi site-directed mu-tagenesis kit. Biotechniques 33:1158 –1160, 1162, 1164 –1165.

60. Schnell S. 2014. Validity of the Michaelis-Menten equation–steady-stateor reactant stationary assumption: that is the question. FEBS J 281:464 – 472. https://doi.org/10.1111/febs.12564.

61. Kou SC, Cherayil BJ, Min W, English BP, Xie XS. 2005. Single-moleculeMichaelis-Menten equations. J Phys Chem B 109:19068 –19081. https://doi.org/10.1021/jp051490q.

Engineering of a (�)-�-Lactamase Applied and Environmental Microbiology

January 2018 Volume 84 Issue 1 e01780-17 aem.asm.org 13

on July 28, 2020 by guesthttp://aem

.asm.org/

Dow

nloaded from