Accepted Manuscript

Title: Caffeic acid O-methyltransferase from Leucaenaleucocephala: Cloning, expression, characterization andmolecular docking analyses

Author: Upendra N. Dwivedi Veda P. Pandey Poonam GuptaSwati Singh Rupinder Singh

PII: S1381-1177(14)00142-8DOI: http://dx.doi.org/doi:10.1016/j.molcatb.2014.04.020Reference: MOLCAB 2946

To appear in: Journal of Molecular Catalysis B: Enzymatic

Received date: 22-7-2013Revised date: 24-4-2014Accepted date: 30-4-2014

Please cite this article as: U.N. Dwivedi, V.P. Pandey, P. Gupta, S. Singh, R. Singh,Caffeic acid O-methyltransferase from Leucaena leucocephala: cloning, expression,characterization and molecular docking analyses, Journal of Molecular Catalysis B:Enzymatic (2014), http://dx.doi.org/10.1016/j.molcatb.2014.04.020

This is a PDF file of an unedited manuscript that has been accepted for publication.As a service to our customers we are providing this early version of the manuscript.The manuscript will undergo copyediting, typesetting, and review of the resulting proofbefore it is published in its final form. Please note that during the production processerrors may be discovered which could affect the content, and all legal disclaimers thatapply to the journal pertain.

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Caffeic acid O-methyltransferase from Leucaena leucocephala: cloning, expression,

characterization and molecular docking analyses

Upendra N. Dwivedi*, Veda P. Pandey, Poonam Gupta, Swati Singh and Rupinder Singh

Department of Biochemistry

Lucknow University

Lucknow – 226007, India

* Corresponding Author

E-mail: upendradwivedi@hotmail.com

Telefax- +91-522- 2740132

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Abstract

Angiospermic caffeic acid O-methyltransferase (COMT) conventionally catalyzes the

methylation of caffeic acid as well as 5-hydroxy ferulic acid to ferulic acid and sinapic acid,

respectively, using S-adenosyl-L-methionine (SAM) as methyl group donor. A cDNA of OMT

from Leucaena leucocephala (LlOMT) was cloned, expressed and purified. The expressed

protein was purified to homogeneity on Ni–NTA agarose column with specific activity of 450

nmoles of ferulic acid formed/min/mg protein. Native molecular weight of the purified enzyme

was found to be 80 kDa and that of subunit molecular weight was found to be 40 kDa,

suggesting the homodimeric nature of the enzyme. The optimum temperature and pH was found

to be 37оC and pH 8.0, respectively. Apparent Km of enzyme for caffeic acid and SAM was

found to be 220 µM and 2.12 µM, respectively. In view of overlapping/metabolic grid concept of

methylation of substrates in phenyl propanoid pathway, an in-silico approach was used to look

into it. Accordingly, the LlOMT protein was modeled and docked with 16 putative substrates

(intermediates of phenyl propanoid/ monolignol biosynthesis pathway). In silico analyses

revealed that the Co-A ester substrates were most favored among those of substrates, analyzed.

Key words: O-methyl transferase; homology modeling; Leucaena leucocephala; molecular

docking; phylogenetic analysis.

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Article Outline

1.0. Introduction

2.0. Experimental

2.1. Plant materials

2.2. Enzyme extraction

2.3. RNA isolation

2.4. Construction and screening of a cDNA library

2.5. Expression and Purification of recombinant LlOMT protein

2.6. Western blot analysis

2.7. Physico-chemical characterization of purified recombinant LlOMT

2.7.1. COMT activity assay

2.7.2. Molecular Weight Determination

2.7.3. Effect of pH and temperature

2.7.4. Determination of Km and Vmax for Caffeic acid and SAM

2.8. In silico analyses of LlOMT

2.8.1. Phylogenetic analysis of LlOMT

2.8.2. Primary, secondary and tertiary structure prediction

2.8.3. Molecular docking Analysis

3.0. Results and Discussion

3.1. Isolation and Characterization of LlOMT cDNA

3.2. Expression and purification of recombinant LlOMT

3.3. Western Blotting Analyses

3.4. Physicochemical characterization of purified recombinant LlOMT

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3.4.1. Molecular Weight Determination

3.4.2. Effect of substrate concentration on the LlOMT activity

3.4.3. Effect of pH on the LlOMT activity

3.4.4. Effect of temperature on the LlOMT activity

3.5. In-silico Analyses of LlOMT

3.5.1. Phylogenetic analysis of LlOMT

3.5.2. Primary, secondary and tertiary structure prediction

3.5.3. Molecular Docking Analyses

Conclusion

Acknowledgements

References

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1.0 Introduction

Methyltransferases (MTs) are ubiquitous enzymes that catalyze the transfer of a methyl

group from S-adenosyl-L-methionine (SAM) to an acceptor substrate, generating O-, N-, S- and

C-methyl derivatives and S-adenosyl homocysteine [1]. Among these, the O-methyltransferases

(OMTs) are involved in the biosynthesis of many plant natural products [2, 3]. Joshi and Chiang

[4] have proposed two classes of OMTs. Class 1 OMTs, low molecular weight (23-27 kDa), are

found to be Mg2+ dependent OMTs and do not accept caffeic acid as substrate whereas, Class 2

OMTs, relatively high molecular weight (38-43 kDa), are independent of Mg2+, and have been

supposed to methylate various substrates including caffeic acid.

Among OMTs, caffeic acid/ 5-hydroxy ferulic acid 3/5-O-methyltransferase (COMT; EC

2.1.1.68) is one of the principal methylating enzymes of phenyl propanoid (PP) pathway that

leads to formation of lignin, flavonoids, phenylpropenes and alkaloids etc. Thus, COMT plays

significant roles in various physiological processes in plant life cycle such as lignification, flavor

generation, fruit ripening etc. [3, 5-6]. On the basis of sequence analyses, it is proposed that

COMTs are encoded by multigene families in many plant species [7]. Classically, in

angiosperms, the COMT is considered as bifunctional enzyme, catalyzing 3-O-methylation of

caffeic acid to ferulic acid and 5-O-methylation of 5-hydroxy ferulic acid to sinapic acid [8].

Based on various in-silico and/or in-vitro experimental studies, reported over more than a

decade, alternative pathways for methylation in the PP pathway have been proposed.

Furthermore, the concepts of “metabolic grid” and “overlapping pathways,” suggested that the

COMT can also methylate various putative substrates/intermediates of PP pathway at the levels

of acids, aldehydes and alcohols [9, 10]. Several OMT cDNA clones have been isolated,

expressed, purified and characterized from a number of sources like Festuca arundinacea,

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Fragaria x ananassa, Oriza sativa, Triticum aestimum, Medicago sativa [11-16].

COMTs differ in their selectivity with respect to the stereochemistry of the methyl

acceptor molecules, as well as the substitution pattern of their phenolic hydroxyl groups [1].

Based on the kinetic properties, alfalfa COMT exhibited its relative activities for all putative

substrates of the PP pathway, in the following order: caffeoyl CoA > caffeoyl alcohol > 5-

hydroxy ferulic acid > caffeoyl aldehyde > 5-hydroxy coniferyl alcohol > 5-hydroxy feruloyl

CoA > 5-hydroxy coniferaldehyde > caffeic acid [15].

Native molecular weight of COMT was reported to be in the range of 60–80 kDa,

suggesting its homodimeric nature [8, 17]. Furthermore, the SDS-PAGE analysis of COMT also

suggested it to be a dimeric enzyme [18] with a subunit molecular weight ranging from 38-43

kDa. Thus, the subunit molecular weight was reported to be 38-43 kDa in tobacco [19], 38 kDa

in pea [20], 41-43 kDa in alfalfa [21], 40 kDa in aspen [8] and 39 kDa in sugarcane [22].

In the present paper, we report gene cloning, recombinant protein expression, purification

and physicochemical characterization of OMT from the stem secondary xylem of Leucaena

leucocephala, a nitrogen fixing tree legume, valued as an excellent source of nutritious forage as

well as a major source of pulpwood for pulp and paper [3]. Furthermore, OMT was modeled and

docked with 16 putative substrates (intermediates of PP pathway) using various Bioinformatical

tools.

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2.0 Experimental

2.1 Plant materials

Leucaena leucocephala cv K29 plants, growing in the garden of Department of Bio-

chemistry, University of Lucknow, India was used as plant material. The young stem, mature

stem, root and leaves of Leucaena leucocephala were collected in liquid nitrogen and used as

plant material for experimental studies.

2.2 Enzyme extraction

One gram each of young stem, old stem, root and leaves tissues of Leucaena

leucocephala were ground in liquid N2 and homogenized in 5 volume (w/v) extraction buffer

containing Tris-HCl (100 mM, pH 7.5), PVPP (0.1 %) and β- mercaptoethanol (20 mM). The

homogenates were centrifuged at 12,000 x g in Sorval RC5C at 4°C for 25 min and supernatents

were used as source of enzyme for western blotting analyses.

2.3 RNA isolation

Total RNA was isolated from old stem secondary xylem of Leucaena leucocephala using

the protocol of Hosein [23]. Poly (A)+ mRNA was purified from the total RNA using oligo-dT

cellulose affinity column chromatography following the protocol of Sambrook and Russel [24].

2.4 Construction and screening of a cDNA library

The cDNA library was prepared using the ZAP Express® cDNA Synthesis Kit and ZAP

Express® cDNA Gigapack® III Gold Cloning Kit from Statagene and subjected to in vivo

excision following the protocol of manufacturer. The prepared cDNA library exhibited pfu of 3.6

x106.

For polymerase chain reaction (PCR) based screening of the in vivo excised cDNA

library of L. leucocephala, OMT specific primers were designed from the conserved regions

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found within various plant OMT protein sequences. The sequences of primers are: forward

primer 5’-CCGACCCATGTCAACGACGAGGAAGCGAACC-3’ and reverse primer 5’-

TTAAGGCTTCTTGAGGAACTCCATGATGTA-3’. PCR amplification of in vivo excised

library was carried out with the thermal parameter of initial denaturation at 94°C for 5 min; 35

cycles of cyclic denaturation at 94°C for 45 sec, annealing at 55°C for 30 sec, extension at 72°C

for 1 min and final extension at 72°C for 5 min in DNA Engine (PTC 200, MJ research; Now

Bio-Rad, USA).

The amplicon was ligated in pGEM T-Easy vector (Promega; Madison, USA) using PCR

based cloning approach and transformed in E. coli DH5α cells. The transformants were screened

out by blue white selection on LB agar plate containing ampicillin (100μg/mL), using the

protocol as described by Sambrook and Russel [24]. The plasmid was isolated from the positive

recombinant colony and sequenced commercially.

The cloned cDNA sequence was analyzed for homology at nucleotide level using

BLASTn tool at NCBI and was translated into amino acid sequence using online Translator tool

on ExPASy (www.ebi.com). Various properties of the deduced LlOMT were investigated using

ProtParam tool of ExPASy (http://expasy.org/tools/protparam.html).

2.5 Expression and Purification of recombinant LlOMT protein

The pGEM T Easy vector having LlOMT clone and pET 28a (+) vector was subjected to

double digestion by restriction enzymes EcoRI and NdeI following the protocol of Sambrook

and Russel [24]. The digested pET 28a(+) vector was subjected to dephosphorylation using calf

intestine alkaline phosphatase (CIAP) in order to avoid self ligation. The reaction mixture,

containing 10x reaction buffer (MBI Fermentas) 2 μl, digested pET 28a(+) vector 10 μl, Calf

Intestine Alkaline Phosphatase (MBI Fermentas) 1 μl (1U/ μl) in a total volume of 20μl was

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incubated at 37○C for 30 min and terminated by heating at 75○C for 10 min in presence of 5 mM

EDTA (pH 8.0). The dephosphorylated vector was recovered after agarose gel electrophoresis

using gel extraction kit of QIAGEN following the protocol of manufacturer.

The LlOMT sequence was ligated in pET 28a(+) vector as described by Sambrook and

Russel [24]. The reaction mixture (10μl) containing 10x T4 DNA ligase buffer (MBI Fermentas)

1 μl, Insert DNA (LlOMT) (100ng) 4 μl, dephosphorylated pET 28a(+) vector (40 ng) 4 μl, T4

DNA ligase (MBI Fermentas) 1 μl (1U) was incubated at 22○C for 1 hour. The ligation product

was used to transform BL21 pLyse S competent cells. The transformed cells were screened on

LB agar plate containing kanamycin (50µg/ml) and plasmid DNA from the screened colonies

was subjected to PCR with gene specific primers to confirm the presence of insert.

Culture of E. coli BL21 (DE3) pLysS cells, harboring LlOMT clone in pET-28a(+), was

grown in LB medium containing kanamycin (50 μg/ml) at 370C with constant shaking at 200

rpm. Culture was grown until their absorbance at 600 nm reached 0.6. At that point, the

expression of recombinant LlOMT was induced by addition of IPTG to a final concentration of 1

mM and the cells were grown for overnight at 37°C with constant shaking at 200 rpm. The cells

were harvested by centrifuging at 1000 x g for 5 min at 4°C in Sigma 5K30 cooling centrifuge

(Sigma Inc., USA). The pellet was washed twice with Tris-HCl, pH 7.5 buffer (100 mM),

resuspended in the same buffer and subjected to two cycles of freeze thaw at -70°C and 4°C,

respectively, with vortexing. The supernatant was collected after centrifuging at 12,000 x g for

15 min at 4°C. An un-induced culture was used as a negative control. The recombinant LlOMT,

having a 6x-histidine tag was subjected to affinity purification using Ni NTA agarose affinity

column chromatography. The bound recombinant protein was eluted using various concentration

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of imidazole (50, 100, 250 and 1000 mM). The eluted protein was dialyzed against 20 mM Tris–

HCl buffer (pH 7.5).

2.6 Western blot analysis

The polyclonal antibody against purified LlOMT was raised in rabbit commercially. The

purified LlOMT as well as the extracts from various tissues of L. leucocephala was subjected to

western blot analysis with the commercially raised polyclonal antibody. The blot was developed

using western blot development kit (Bangalore GeNei), following the protocol of manufacturer

with 1:500 dilution of OMT antibody.

2.7 Physico-chemical characterization of purified recombinant LlOMT

2.7.1 OMT activity assay

OMT enzyme activity was determined as described by Bugos et al., [8] with slight

modifications using caffeic acid as substrate. The reaction mixture (200 μl) comprised of 50 mM

Tris HCl (pH 8.0), 1 mM caffeic acid and 100 μl enzyme solution. After preincubation for 5 min

at 37°C, 1 μl of 100 nmol of a mixture of hot and cold S-adenosyl-L-methionine (SAM) was

added and the reaction was incubated at 37°C for 30 min. Hot and cold mixture of SAM

comprised of 100 μl of S-adenosyl-L-[methyl-14C] methionine (SAM-14Me; 2.15 GBq mol-1; 54

mCi mmol-1; Amersham Pharmacia) and 4.9 mg SAM.HSO4. The reaction was terminated by

the addition of 20 μl 2M HCl. Labelled ferulic acid, thus formed, was extracted in 1 ml of

diethylether (Et2O) by vigorous shaking and the two phases were separated by centrifugation at

10000 x g for 10 min at room temperature. The tubes were placed at –20°C for 1 hr to freeze the

lower aqueous phase and the upper Et2O phase was transferred to a scintillation vial.

Radioactivity was determined by LKB Rack Beta liquid scintillation Counter after addition of 5

ml of scintillation cocktail. Control assay contained no caffeic acid. For the determination of

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COMT activity, the counts per min (cpm) for controls of each sample were subtracted from the

respective experimental values. OMT activity was expressed in units of nmol of ferulic acid

formed min-1 g-1 fresh weight (104 cpm ≡ 53 nmoles of ferulic acid). Specific activity was

expressed in terms of nmoles of ferulic acid formed/min /mg protein.

2.7.2 Molecular Weight Determination

The native molecular weight of purified LlOMT was determined using sephadex G-200

column chromatography. The column void volume (Vo) was determined by eluting Blue

Dextran. Catalase (240kDa), Alcohol Dehydrogenase (150kDa), Phosphorylase B (97.4kDa),

Bovine Serum Albumin (67kDa) and Lysozyme (14.3kDa) were used as standard protein for

caliberating the column. Each standard protein (1 mg per ml) was applied on to the column and

protein in column effluent was monitored by Bradford’s method [25]. The elution volume (Ve)

of each standard protein as well as purified LlOMT was calculated. The molecular weight of the

LlOMT protein was calculated from a calibration curve, where log of the molecular weights of

the standards were plotted against the ratio of the elution volumes of the standards and the void

volume of the column.

For the determination of subunit size and composition of purified LlOMT, SDS-PAGE

was done using 10% resolving gel with 3% stacking gel as described by Lamelli [26].

2.7.3 Effect of pH and temperature

Effect of pH on the purified LlOMT activity was investigated in pH ranging from 5.0 to

9.0 using sodium phosphate buffer for pH 5.0-6.5 and Tris–HCl buffer for pH 7.0 - 9.0.

Effect of temperature on the activity of recombinant LlOMT was investigated by

incubating the reaction mixture at different temperatures (25-700C) and determining the enzyme

action.

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2.7.4 Determination of Km and Vmax for Caffeic acid and SAM

Km for the substrate caffeic acid was determined by varying the final concentration of

caffeic acid between 30 µM to 2 mM, while SAM concentration was kept at a constant level of

0.5 µM. Similarly, Km for the SAM was determined by varying the concentration of SAM

between 0.01 µM to 15 µM, while caffeic acid was kept at a constant level of 1mM. Km and

Vmax values were calculated using Lineweaver-Burk plots.

2.8 In silico analyses of LlOMT

2.8.1 Phylogenetic analysis of LlOMT

To find out the evolutionary relatedness of the LlOMT, phylogenetic tree was

constructed, using Neighbor joining (NJ) method with the MEGA 4.1 software.

2.8.2 Primary, secondary and tertiary structure prediction

The primary structure analysis of LlOMT was performed using ProtParam tool of

ExPASy (www.ebi.com). The secondary structure prediction of the LlOMT protein was done

using SOPMA tool (http://npsa-pbil.ibcp.fr/cgibin/ secpred_sopma.pl). Motif analyses were also

performed for identifying conserved regions using ClustalW tool. In order to predict tertiary

structure of LlOMT, a template was selected through BLAST Search (DS-server) using

Discovery Studio 3.5 software. Several initial models were constructed, using Discovery Studio

and the one with minimum DOPE score was retained. Finally, the model was further validated

by various in-silico tools including PROCHECK, ERRAT and ProQ.

2.8.3 Molecular docking Analysis

Various metabolic intermediates of PP pathway, namely caffeic acid, sinapic acid, ferulic

acid, 5-hydroxyferulic acid, their CoA esters, and coniferyl and caffeoyl alcohols, their

aldehydes, 5-hydroxy coniferyl aldehyde, 5-hydroxy coniferyl alcohol, SAM, and SAH were

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considered as putative substrates for OMT and used as ligands for docking. The mol files of

these ligands were retrieved from the Chemspider and Pubchem databases. Preparation and

minimization of the ligands were done by applying CHARMm force field. The protein structure

(modeled LlOMT) was also prepared by applying CHARMm force field. The so prepared protein

model was subjected to docking with various ligands using CDOCKER tool of Discovery Studio

3.5.

3.0 Results and Discussion

3.1 Isolation and Characterization of LlOMT cDNA

LlOMT cDNA was cloned by PCR based screening of Leucaena leucocephala cDNA

library and commercially sequenced. The 1098 bp cDNA clone (Accession no. EF546435) on

BLAST analysis showed 92% identity with the COMT from Acacia mangium x Acacia

auriculiformis and 80% with Medicago sativa, authenticating the clone to be as COMT (data not

shown). Deduced amino acid sequence of the LlOMT clone consisted of 365 amino acid residues.

At amino acid level the LlOMT exhibited a high degree of homology (96%), with those of

Acacia mangium x Acacia auriculiformis, belonging to same family leguminosae as Leucaena

leucocephala (data not shown).

3.2 Expression and purification of recombinant LlOMT

The LlOMT gene in pGEM T-Easy vector (Promega) was subcloned in pET-28a (+)

vector and transformed into BL21 (DE3) pLysS cells of E. coli. The expressed and active

recombinant LlOMT was purified through Ni-NTA agarose affinity column chromatography.

The recombinant LlOMT was purified to homogeneity with 250 mM imidazole (Fig 1A). The

purified LlOMT exhibited a specific activity of 450 nmoles of ferulic acid formed/min/mg

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protein. The single band on SDS-PAGE (Fig 1 A) was suggestive of the homogenous preparation

of LlOMT.

3.3 Western Blotting Analyses

The specificity of the polyclonal antibody raised against purified recombinant LlOMT

was checked and it was found to hybridize strongly with that of induced protein (Fig1B).

Furthermore, the cross-reactivity of polyclonal antibody against purified recombinant LlOMT to

that of naturally occurring COMT was also established by performing western blot analysis of

COMT isolated from young stem, old stem, root and leaves of Leucaena leucocephala. The

results are presented in Fig 1C. It is noteworthy that the extent of hybridization was in

accordance with the occurrence of levels of OMT in these tissues, with old stem reacting most

strongly followed by young stem, root and that of leaf [8].

3.4 Physicochemical characterization of purified recombinant LlOMT

3.4.1 Molecular Weight Determination

Based on SDS-PAGE analyses (Fig 1A), the purified LlOMT protein exhibited a subunit

molecular weight of 40 kDa, suggesting it to be a high molecular weight, magnesium

independent type 2 OMT as classified by Joshi and Chiang [4]. Similar to our finding, Festuca

arundinacea, Strawberry, Populus tremuloides, Medicago sativa OMTs are reported to be of

size 40 kDa [11-12, 27-28] whereas, that of Oryza sativa and Triticun aestimum OMT are 41

kDa and 42.3 kDa, respectively [13-14].

For determination of the native molecular weight of the purified LlOMT protein, gel

filtration chromatography was performed using standard proteins. Results are shown in Fig 2.

Native molecular weight of the purified LlOMT was found to be ~80 kDa suggesting a

homodimeric structure of LlOMT, consisting of two subunits of size 40 kDa. Similarly,

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Strawberry and Ruta graveolens OMTs are reported to be homodimeric protein with native

molecular weight of 80 and 84 kDa, respectively [12, 29]. Based on crystallographic analysis,

Zubieta et al., [16] has also reported Medicago sativa OMT as a homodimeric protein.

3.4.2 Effect of substrate concentration on the LlOMT activity

Effect of phenolic substrate caffeic acid and methyl group donar SAM on the activity of

LlOMT was investigated by varying the concentration of one of each and keeping the

concentration of other as fixed. The Km and Vmax were determined using double reciprocal

plots (Fig 3.). The Km and Vmax for caffeic acid was found to be 220 µM and 625 nmoles of

ferulic acid formed/min/mg protein, respectively, while that of, for SAM was found to be 2.12

µM and 2400 nmoles of ferulic acid formed/min/mg protein, respectively.

In literature, much lower Km for caffeic acid has been reported. For example, the OMTs

from Festuca arundinacea, Mesembryanthemum crystallinum, Oryza sativa, Triticum and

Medicago show Km value of 49.74 µM, 44 µM, 69 µM, 68.75 µM and 59.5 µM, respectively

[11, 30, 13- 15]. A similar Km for SAM (2 µM) has been reported for Ruta gravelons OMT

[29]. However, a higher Km for SAM (51 µM) has also been reported for Oryza sativa [13].

3.4.3 Effect of pH on the LlOMT activity

The effect of pH on purified LlOMT was investigated. The purified enzyme exhibited

optimum pH of 8.0 and was found to be fairly stable at the pH range of 6.5-8.5 (data not shown).

The enzyme lost about 55 % and 42% of its activity at pH 5.0 and pH 9.0, respectively. The pH

optima of 7.5 have been reported for OMT from Oryza sativa [13], Ruta graveolens [29] and

Festuca arundinacea [11]. While a pH optimum of 8.5 has been reported for OMT from

Strawberry [12].

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3.4.4 Effect of temperature on the LlOMT activity

The effect of temperature on purified LlOMT activity was determined. The purified

enzyme exhibited broad temperature optima between 25-45°C with maximum activity at 37°C.

Beyond 45°C LlOMT activity declined rapidly with a complete loss of activity at 70oC. Most of

the OMTs reported in literature have temperature optima in the range of 35-37°C. Thus, OMT

from Ruta gravelons, Strawberry and Oryza sativa exhibited temperature optima of 36°C, 37 °C

and 35 °C, respectively [29, 12-13], whereas, OMT from Vitis vinifera, has been reported to

possess temperature optimum in the range of 45-50°C [31].

3.5 In-silico Analyses of LlOMT

3.5.1 Phylogenetic analysis of LlOMT

In order to study the evolutionary relationship of LlOMT, the phylogenetic analysis was

done. The results are shown in Fig 4. The data presented in Fig 4, revealed a divergent evolution

for the LlOMT. It is noteworthy that the COMTs were found to be clustered into two major

groups. Group A represented angiospermic COMTs, whereas group B represented COMT

sequences from gymnosperms. Group A COMT could further be classified into classes I and II,

with class I representing sequences from dicots and class II representing monocot COMTs. This

evolution in OMT was supplemented to the alpha taxonomical division of the taxa into

gymnosperm and angiosperm and further division of angiosperms into monocots and dicots.

LlOMT was found to be clustered with other plants like Acacia and Medicago from leguminosae

family and assembled in same group. LlOMT possesses a position with all dicots and showing a

distance from monocots and the gymnosperm. Thalictum tuberosum is a eudicot which possess

an intermediate position between dicots and monocots. With reference to evolution, monocot

COMTs shows close relation with gymnosperms COMT protein sequences.

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3.5.2 Primary, secondary and tertiary structure prediction

The primary structure analysis using ProtParam tool revealed a predicted molecular

weight of 39839.39 kDa with theoretical pI of 5.60. In addition, the protein has 42 negatively

charged residues and 33 positively charged residues. Secondary structure predicted through

SOPMA (http://npsa-pbil.ibcp.fr/cgi-bin/secpred_sopma.pl) exhibited 47.95% α-helices, 21.00%

β sheets and 30.68% random coils. Motif analyses revealed total seven motifs (Fig 5 A), out of

which six motifs (no. 2-7) were localized toward carboxy terminus and remaining one motif (no.

1) was localized towards N terminus. The protein exhibited four caffeic acid binding motifs

namely, motif 1(LDRMMRLL) from amino acids 77 to 84 (only conserved motifs towards the

amino terminus), motif 3 (INGINFDLPHVI) from 225 to 236, motif 4 (PGVEHVGGDMF) from

243 to 253 and motif 7 (GGKERTEKEFEAL) from 326 to 338. The three SAM motifs namely,

motif 2 (LVDVGGGTG), motif 5 (VPKADAVFMKWI) and motif 6 (ALPENGKVIVAECILP)

were localized from amino acids 204-212, 256-267 and 286-301, respectively. The motif

prediction analyses are also in agreement with earlier reports [4, 27].

The tertiary structure was predicted by Discovery Studio 3.5 software using Medicago

sativa COMT (PDB ID: 1kyw) as template. The accepted model with minimum DOPE score (-

27999.525391) is presented in Fig 5B. The predicted 3D structure of LlOMT was validated using

PROCHECK, ERRAT, ProQ tools (Table 1) and Ramachandran plot (Fig 5C). PROCHECK was

used to check the stereochemical quality of a protein structure by analyzing residue-by-residue

geometry through Ramachandran plot. LG score in ProQ is -log of P-value and LG scores >1.5 is

considered as correct model. Thus, the LG score of modeled LlOMT was found to be >2. The

overall quality factor in the ERRAT tool, is used to find out the reliability of the generated model

and expressed in terms of percentage and represent the percentage of the modeled protein for

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which the calculated error value falls below the 95% rejection limit. Hence, based on all the

above validation tools, the modeled LlOMT was found to be reliable. The predicted model

showed that the protein consisted of two domains, one small domain and one large domain. The

small domain is found at the N-terminus of a variety of plant O-methyltransferases and has been

shown to mediate dimerisation of these proteins [16].

3.5.3 Molecular Docking Analyses

The modeled LlOMT was docked with 16 substrates (metabolic intermediates of

monolignol biosynthesis pathway) and results are presented in Table 2. The -CDOCKER energy

obtained from docking with various substrates to LlOMT decreased in the following order: thiol

ester substrates > free acid substrates > alcoholic and aldehyde substrates, suggesting the best

binding for thiol ester substrates with LlOMT. In agreement of our in-silico docking studies,

Maury et al., [32] have suggested that tobacco COMT efficiently interacted with CoA ester

substrates and catalyzed the methylation of these substrates more efficiently than those of free

acids. Similarly, Parvathi et al. [15] have also reported alfalfa COMT catalyzed methylation of a

number of putative substrates of monolignol biosynthesis pathway, with higher enzyme activities

for CoA ester substrates than that of free acids. Furthermore, based on in silico analyses on

substrate specificity of COMT, Naaz et al., [33] have recently reported that COMT showed

higher affinity towards CoA ester substrates than that of free acids. A 3D representation for

binding of 5-hydroxyferuloyl CoA (most favored substrate) is shown in Fig. 5B.

A comparison of interacting residues of LlOMT with all the 16 putative substrates

revealed that ARG171, VAL175, VAL254, PRO257, ALA286, LEU300, PRO304, LEU308,

ALA342, GLY343, PHE344 are the residues that interact with almost all the substrates (Table

2). A 2D representation for interacting residues of LlOMT with 5-hydroxy feruloyl CoA (most

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favored substrate) is shown in Fig. 6. The binding pocket of COMT containing LEU, PHE and

ALA were reported to interact with ferulic acid and 5-hydroxyconiferaldehyde [16].

Furthermore, on the basis of a crystallographic study of alfalfa COMT, Zubieta et al. [16] have

reported that the PHE is interacted with SAM. Their findings are in agreement with our docking

analysis data. Based on in-vitro wet laboratory studies, it has been shown that the SAM binding

sites of various O-methyltransferases are conserved. Thus, the residues namely LYS and ASP of

rice COMT are reported to interact with SAM [16, 34-35]. Similarly, in our study both the

residues were also found to interact with SAM.

Though, COMTs have traditionally been thought to catalyze the methylation of caffeic

acid [36] but, in the past ten years or so with newer studies, based on wet lab and in-silico

analyses, it has been shown that the COMTs are multifunctional enzymes catalyzing the

methylation of a variety of phenolic substrates in phenylpropanoid pathway as well as

alkaloid/flavanoid pathways [33, 37]. In case of LlOMT, a high Km for caffeic acid

(experimental findings) as well as higher –CDOCKER energy (in-silico prediction) is suggestive

of the fact that this OMT might also utilize a number of other phenolic substrates for

methylation. Thus, from the data presented, it was concluded that the Leucaena leucocephala

OMT could be considered as a member of class 2 OMT, a high molecular weight protein and has

potential to catalyze methylation of various putative substrates in addition to conventional

substrates of phenyl propanoid pathway.

Conclusion:

Caffeic acid/ 5-hydroxy ferulic acid 3/5-O-methyltransferase is one of the principal

methylating enzymes involved in lignin biosynthesis in angiosperm. In the present paper, a

cDNA of OMT from Leucaena leucocephala (LlOMT) was cloned, expressed and the

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recombinant protein was purified and characterized. The purified LlOMT was found to be a

homodimeric protein of 80 kDa size. The optimum temperature and pH was found to be 37оC

and pH 8.0, respectively. The LlOMT protein was modeled and docked with 16 putative

substrates (intermediates of phenyl propanoid/ monolignol biosynthesis pathway). The docking

analyses revealed that the Co-A ester substrates were most favored among those of acidic,

alcoholic and aldehydic substrates. Findings of the present study in a long way are helpful in

developing transgenic plants of desired traits by metabolic engineering approaches for their

improved applicability with regards to paper industry, forage digestibility and bioenergy

production.

Acknowledgments

Financial assistance from the Department of Biotechnology (DBT), Govt. of India, New

Delhi (in the form of JRF to PG) is gratefully acknowledged. We are also thankful to Department

of Higher Education, Govt. of U.P., under the Center of Excellence in Bioinformatics

programme, Department of Biotechnology (DBT), Govt. of India, New Delhi under the BIF

programme and Department of Science & Technology under Promotion of University Research

and Scientific Excellence (DST-PURSE) programme.

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Figure Captions:

Fig. 1: A. SDS-PAGE profile of LlOMT during various stages of purification using Ni NTA

agarose column. Lane 1, protein molecular weight marker; lane 2: crude protein; lane 3:

wash through; lane 4: purified fraction eluted with 250 mM imidazole.

B. Western blot analysis: Lane 1: un-induced (without IPTG); lanes 2: induced with 1

mM IPTG. C. Western blot analysis showing the cross-reactivity of COMT in

various tissues of Leucaena leucocephala. Lane 1: young stem, Lane 2: old stem,

Lane 3: root and Lane 4: leaves.

Fig. 2: Molecular weight determination of purified LlOMT by gel filtration chromatography.

Fig. 3: Lineweaver Burk and Mechaelis-Mentan plots for the effect of various concentration of

caffeic acid (A and B) and SAM (C and D), respectively.

Fig. 4: Phylogenetic tree for COMTs from 37 plant sources based on homologous groups

showing evolutionary relationships with LlOMT. Numbers left of the nodes are

bootstrap values indicating frequencies of respective furcations found in 1000

replications of subset tree calculations.

Fig. 5: A. Conserved motif analyses in LlOMT. Motif 1 (77-84); SAM motif 2 (204-212); motif

3 (225-236); motif 4 (243-253); SAM motif 5 (256-267); SAM motif 6 (286-301) and

motif 7 (326 -338).

B. 3D representation showing the binding of 5-hydroxyferuloyl CoA to LlOMT

C. Ramachandran Plot.

Fig.6. 2D view showing the interacting residues of LlOMT with 5-hydroxyferuloyl CoA

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Table 1. Validation of predicted model of LlCOMT using various tools

Tools Parameters Value

CDOCKER DOPE score -27999.525391

Core region 88.1%

Additional Allowed region 8.9%

Generously allowed regions 2.0%

PROCHECK

(Ramachandran

Plot)

Disallowed regions 1.0%

ProQ Predicted LG Score 2.031

ERRAT Overall Quality factor 83.626

 

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Table 2: Summary of results of docking of sixteen putative substrates with LlCOMT depicting

the specific amino acid residues interacting with the putative substrates at the enzyme

active site. Additional residues, namely ARG171, VAL175, VAL254, PRO257,

ALA286, LEU300, PRO304, LEU308, ALA342, GLY343, PHE344 were found common

to interact with these putative substrates at the enzyme active site.

S.

No.

Substrate -CDOCHER

Energy

(KJ/mol)

Specific Interacting Residues

1 Caffeic Acid 25.8933 PHE253, ASP285, PRO301, ASP305, ASP317

2 Ferulic Acid 25.3801 PHE253, PRO301, ASP305, ASP317

3 Sinapic Acid 22.4859 ASN118, GLU119, ILE124, LYS168, SER303,

SER307

4 5-hydroxy-ferulic

Acid

27.9953 PHE253, LEU300, PRO301, ASP305, ASP317

5 5-hydroxy Coniferyl

Aldehyde

24.8065 PHE253, PRO301, ASP317

6 5-hydroxy Coniferyl

Alcohol

18.8454 LYS168, ASP285, PRO304, SER307, ASP317

7 Caffeoyl Aldehyde 23.7177 LEU115, ASN118, GLU119, LYS168, ASP285,

SER307

8 Caffeoyl Alcohol 16.9791 LEU115, GLU119, LYS168, ASP285, SER307

9 Coniferyl Aldehyde 22.3948 PHE253, PRO301, ASP317

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10 Coniferyl Acohol 15.2578 VAL175, ASP285, PRO301, ALA342

11 5-hydroxyferuloyl

CoA

97.0179 ILE102, ALA112, LEU115, ASN118, GLU119,

ILE124, GLU164, LYS168, ASP169, PHE172,

TYR195, THR196, THR201, SER255, LYS258,

SER307, SER309, LYS311, HIS315

12 Sinpoyl CoA 94.5323 ALA112, LEU115, TYR165, LYS168, ASP169,

PHE172, LYS174, PHE198, GLU199, LEU201,

PHE253, ASP285, PRO301, PRO304, HIS315

13 Caffeoyl CoA 91.0872 ALA112, LEU115, TYR165, LYS168, ASP169,

PHE172, LYS174, PHE198, GLU199, LEU201,

PHE253, ASP285, PRO301, HIS315

14 Feruloyl CoA 88.8177 MET24, GLU103, ALA112, LEU115, VAL116,

GLU119, TYR165, LYS168, ASP169, PHE172,

TYR195, THR196, PHE198, THR202, LEU211,

PHE253, SER255, LYS258, ASP285, SER307,

LYS311, HIS315

15 SAM

11.1613 LEU115, GLU119, LYS168, PHE253, ASP285,

PRO301, SER307, ASP317

16 SAH

17.5829 ALA112, LEU115, VAL116, ASN118,

GLU119, LYS168, LYS258, SER303, SER307,

HIS315

 

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Highlights 

cDNA of  COMT (1098bp) from Leucaena leucocephala stem was cloned. 

Recombinant LlCOMT was expressed, purified and characterized.  

Based on molecular docking, thiol esters were found to be best substrates. 

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Graphical abstract

3D structure of modelled LlCOMT docked with methyl donar (SAM ) and best putative substrate (5-hydroxy feruloyl CoA) as ligands.

*Graphical Abstract (for review)

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Figure 1

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Figure 2

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Figure 3

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Figure 4

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Figure 5

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Figure 6