structure-function analyses and molecular modeling of caffeic acid-o-methyltransferase and...

Structure–function analyses and molecular

modeling of caffeic acid-O-methyltransferase

and caffeoyl-CoA-O-methyltransferase:

Revisiting the basis of alternate methylation

pathways during monolignol biosynthesis

Huma Naaz

Veda P. Pandey

Swati Singh

Upendra N. Dwivedi∗

Department of Biochemistry, Bioinformatics Infrastructure Facility, Centreof Excellence in Bioinformatics, University of Lucknow, Lucknow, U.P., India

Abstract

Ten protein sequences, each of caffeicacid-O-methyltransferase (COMT) and caffeoyl-coenzymeA-O-methyltransferase (CCoAOMT), catalyzing methylation ofprecursors of monolignol from selected dicots and monocotshave been analyzed and compared on the basis of their aminoacid sequence, motifs/domains, three-dimensional (3D)structure, and substrate binding. The isoelectric points of allthe COMT and CCoAOMT sequences analyzed were found tovary in the pH range of 5 to 6. Molecular weight analysessuggested CCoAOMT to be smaller monomeric proteins(27–29 kDa) as compared with those of COMTs (39–40 kDa),which were dimeric. On the basis of phylogenetic analysis,

COMT and CCoAOMT were clustered into two major groups,each of which could be further divided into two subgroups ofmonocots and dicots. Modeling and superimposition of COMTand CCoAOMT sequences of alfalfa (Medicago sativa) revealedthat both were quite different at the 3D levels, although theyhad similarity in the core region. Molecular docking of 16putative substrates (intermediates of monolignol biosynthesispathway) revealed that both enzymes interact with all 16substrates in a similar manner, with thiol esters being the mostpotent and binding of these putative substrates to CCoAOMTbeing more efficient. C© 2013 International Union of Biochemistry andMolecular Biology, Inc. Volume 60, Number 2, Pages 170–189, 2013

Keywords: COMT, CCoAOMT, homology modeling, molecular docking,phylogenetic analysis, substrates specificity

1. IntroductionLignin, a phenolic heteropolymer, is present in the cell wallof plants and provides rigidity, strength, and resistance tochemical, physical, as well as biological attacks. Lignin, which

Abbreviations: SAM, S-adenosyl methionine; SAH, S-adenosylhomocysteine; MD, Modeller; DS, Discovery Studio; ProSA, proteinstructure prediction; ProQ, protein quality predictor; SAVES, structuralanalysis and verification server.∗Address for correspondence: Upendra N. Dwivedi, PhD, Department ofBiochemistry, Bioinformatics Infrastructure Facility, Centre of Excellence inBioinformatics, University of Lucknow, Lucknow 226007, Uttar Pradesh,India. Tel.: +91 522 2740132; Fax: +91 522 2740132; e-mail:[email protected] 15 August 2012; accepted 4 December 2012DOI: 10.1002/bab.1075Published online 18 April 2013 in Wiley Online Library(wileyonlinelibrary.com)

composes an average of 25% of the plant biomass, is consideredas one of the greatest obstacles to the optimal utilization ofthe plant biomass for various purposes such as paper manu-facturing, production of highly palatable forage, and bagasseutilization. Chemically, lignin is made up of mainly three kindsof monolignols, namely p-coumaryl alcohol, sinapyl alcohol,and coniferyl alcohol. These monolignols are synthesizedvia phenyl propanoid pathway, which starts with deamina-tion of phenylalanine, followed by successive hydroxylations,methylations, thiol ester formation, and two reduction re-actions leading to the formation of the three major kinds ofmonolignols. As far as methylation of the intermediates inthe lignin biosynthesis pathway is concerned, classically ittakes place at the level of free acids by the enzyme caffeicacid/5-hydroxyferulic acid 3/5-O-methyltransferases (or caf-feic acid-O-methyltransferase); EC 2.1.1.68 (COMT). Thus,COMT is capable of S-adenosyl methionine (SAM)-dependentmethylation of caffeic acid at the 3-hydroxy position to yieldferulic acid and also of 5-hydroxyferulic acid at the 5-hydroxy

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position to yield sinapic acid [1]. Ferulic acid and sinapic acidthen could be converted, successively, to their related thiolesters, aldehydes, and alcohols, yielding the coniferyl (guaiacylor G) and sinapyl (syringyl or S) monolignols, respectively. Thethree monolignols (p-coumaryl alcohol, sinapyl alcohol, andconiferyl alcohol) vary only by the 3- and 5-methoxyl groups.Enzymatic reactions controlling the methylation of the 3- and5-hydroxyls of monolignol precursors are important to de-termine the lignin composition [2]. On the basis of a numberof biochemical and transgenic studies carried out over morethan a decade, alternative pathways for methylation of ligninprecursors have been proposed. According to these so-calledconcepts of “metabolic grid” and “overlapping pathways,”COMT can also methylate substrates at the levels of aldehydesand alcohols, whereas another SAM-dependent methylating en-zyme, namely caffeoyl-coenzyme A/5-hydroxyferuloyl-CoA 3/5-O-methyltransferases [or caffeoyl-CoA-O-methyltransferase](EC 2.1.1.104) (CCoAOMT), can methylate the CoA esters of 3-hydroxy and 5-hydroxy acids [3,4]. These studies have indicatedthat the methylation pathways in monolignol biosynthesis aremuch more complicated than those reported previously [3,5,6].

In view of alternative methylation pathways reported overmore than a decade, based on in vivo/in vitro wet laboratory ex-perimental data, it has been suggested that both COMTs as wellas CCoAOMTs can efficiently catalyze the methylation of sub-strates, which are traditionally not considered as their normalsubstrates [7, 8]. Thus, investigation of tobacco COMT to cat-alyze methylation of various nontraditional substrates, namelycaffeoyl and 5-hydroxyferuloyl esters, aldehydes, as well asalcoholic substrates, revealed efficient catalysis (based onVmax/Km), thereby suggesting that COMT-mediated syringyl unitsynthesis can be operated at the levels of free acids, CoA esters,aldehydes, or alcohols [9]. Furthermore, Parvathi et al. [10],in alfalfa (Medicago sativa) COMT, have investigated COMT-catalyzed methylation with a number of potential substrates ofmonolignol biosynthesis pathway; their results reveal activitiesin the order caffeoyl-CoA > caffeoyl alcohol > 5-hydroxyferulicacid > caffeoyl aldehyde > 5-hydroxyconiferyl alcohol > 5-hydroxyferuloyl CoA > 5-hydroxyconiferaldehyde > caffeicacid. Using antisense downregulated transgenic plants harbor-ing CCoAOMT and COMT gene constructs, they have furtherprovided evidence for an alternative methylation pathway in-volving caffeoyl aldehydes, caffeoyl alcohols, or caffeoyl-CoAesters by COMT. However, there is still ambiguity with regardto substrate affinities and efficiency of catalysis for COMTs andCCoAOMTs. Therefore, this needs further validation.

On the basis of sequence analyses, it is proposed thatCOMT and CCoAOMT belong to two distinct families of O-methyltransferases and are encoded by multigene families inmany plant species [11]. COMTs on an average are made upof about 350 amino acids and have an N-terminal domain(dimerization domain) that participates in homodimerizationand do not require metal ions for activity. As compared withCOMTs, CCoAOMTs are relatively smaller proteins with anaverage of about 220 amino acids and without an N-terminal

dimerization domain and require metal ions for activity. PlantCOMTs in general have been suggested to be dimeric, whereasthose of CCoAOMT are suggested to be monomeric in solutionfor catalysis [5,12,13].

At least three domains have been reported in COMTs,namely AdoMet domain, methyltransferase, and a dimerizationdomain, which are involved in SAM/S-adenosyl homocysteine(SAH) binding, phenolic substrates binding, and dimer forma-tion, respectively. Thus, in the case of alfalfa COMT, Zubietaet al. [5] reported a large C-terminal catalytic domain involvedin SAM/SAH and phenolic substrate binding, whereas a smallerN-terminal dimerization domain was involved in dimer for-mation. Comparison of the CCoAOMT structures with otherwell-known OMT structures exhibits a high level of structuralsimilarity in the SAM binding region of methyltransferases, sug-gesting that the SAM binding protein fold remained conservedduring evolution [14].

In the present paper, 10 each of COMT and CCoAOMT se-quences from selected dicots and monocots have been analyzedand compared for parameters like molecular weights, isoelec-tric point (pI), domain analysis, their cellular localization, thephylogenetic relationship, and substrate specificity. The COMTand CCoAOMT sequences of M. sativa were selected for three-dimensional (3D) structural modeling and docking analyses,with intermediates of monolignol biosynthesis pathway as 16putative substrates in order to find out the efficiency and affin-ity of these substrates toward binding to COMT and CCoAOMTand catalysis. Results of molecular docking analyses have beendiscussed in light of in vivo/in vitro wet laboratory experimen-tal data to throw light on the newer roles of these enzymesin methylation of these intermediates (putative substrates) ofmonolignol biosynthesis pathway.

2. Materials and Methods2.1. Sequence analysesMultiple sequence alignment of 10 sequences each ofCOMT and CCoAOMT from various plant sources wasdone using clustalW tool of EBI (Hinxton, CB, UK)(http://www.ebi.ac.uk/Tools/msa/clustalw2/). A phylogenetictree for these sequences was produced by MEGA (ASU,Tempe, AZ, USA) software to analyze the sequence con-servation. Theoretical pI and molecular weight of COMTsand CCoAOMTs were calculated using PI/MW tool of ExPASy(SIB, Geneva, Switzerland) (http://web.expasy.org/protparam/).The soluble and membrane protein prediction by an-alyzing transmembrane region was done with SOSUI(SIB, Geneva, Switzerland) server (http://bp.nuap.nagoya-u.ac.jp/sosui/). Domain prediction was done with thehelp of the CDD tool of NCBI (NIH, Bethesda, MD, USA)(http://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi).

2.2. Preparation of protein structuresThe 3D structures of COMT and CCoAOMT of M. sativa wereconstructed using Modeller (MD) 9v9 (UCSF, San Francisco, CA,

Biotechnology and Applied Biochemistry 171

Biotechnology andApplied Biochemistry

USA) [15, 16] as well as Discovery Studio 3.1 (DS) (Accelrys,San Diego, CA, USA). Stereochemical validation of the gen-erated models was done using PROCHECK of the structuralanalysis and verification server (SAVES) (UCLA, Los Ange-les, CA, USA) (http://nihserver.mbi.ucla.edu/SAVES/), ERRAT(UCLA, Los Angeles, CA, USA), protein structure prediction(ProSA) (USB, Hellbrunnerstrasse, SB, Austria), and the proteinquality predictor (ProQ) (SBC, Solna, Sweden) tools [17–19].Validated models were subsequently submitted to ProteinModel Database as theoretical models, and were assignedidentity numbers PM0078258 and PM0078259 for COMT andCCoAOMT, respectively.

2.3. Docking studies2.3.1. Preparation of the target and ligands for dockingThe binding pockets and active sites of COMT and CCoAOMTwere recognized by Computed Atlas of Surface Topogra-phy of Proteins (CASTp) server (UIC, Chicago, IL, USA)(http://sts.bioengr.uic.edu/castp/) [20]. The largest cavitiespresent on enzymes were selected for docking the ligands. Themol files of various ligands were retrieved from the Pubchemdatabases of NCBI [21] and these mol files were converted intoPDB files with the help of an open babel converter. Moleculeswere docked at the binding sites by Autodock 4.0 (SRI, La Jolla,CA, USA) [22] as well as by DS.

The binding region for the docking study by Autodock wasdefined at a 0.375 A radius sphere centered on the active site. Agrid box of dimensions 50 × 50 × 50 A3 was constructed aroundthe binding site, based on the cocrystallized ligand. Ten geneticalgorithm (GA) runs were performed for each compound, and10 ligand bumps were allowed in an attempt to account formutual ligand/target fit. Each of the GA runs was performedon a population of 150 individuals. The rates of mutation andcrossover were set to 0.02 and 0.8, respectively. The valueof binding energy was used to rank the docking positions ofthe molecules. The clusters with lowest binding energy wereselected.

Docking was also done with the help of CDOCKER of DS.The binding site was detected by the “define and edit bindingsite” tool, and the binding site was selected on the basis ofreceptor cavities. The random conformations were set to 10with 1,000 dynamic steps; CHARMm force field was used tominimize the structures. The value of CDOCKER energy wasused to rank the docked substrates; the pose with minimumCDOCKER energy was selected to be the best one. Dockinganalysis of all the 16 putative substrates was also done withLIBDOCK of DS and the results were in the form of scores,and the pose with highest score was selected as the best;results of LIBDOCK were in agreement with the CDOCKERenergies.

2.3.2. Enzyme–substrate interactionsDocking interactions between enzymes and putative substrateswere analyzed with the help of the “analyze complexes” optionof CDOCKER tool of DS.

2.4. Superimposition of modeled COMT and CCoAOMTstructuresModeled COMT and CCoAOMT structures were superimposedusing Chimera (UCSF, San Francisco, CA, USA) as well as DS.

3. Results and Discussion3.1. Sequence analyses of COMT and CCoAOMTProtein sequences encoding COMTs and CCoAOMTs from 10common plant sources were analyzed and compared for theirtheoretical pIs, molecular weights, domain prediction, andtransmembrane regions. Results are presented in Table 1. ThepIs of all the COMT and CCoAOMT sequences analyzed werefound to vary in the pH range of 5 to 6, suggesting that all ofthem are acidic in nature. Molecular weights of COMTs werefound in the range of 39–40 kDa, whereas those of CCoAOMTswere in the range of 27–29 kDa, suggesting CCoAOMT tobe smaller proteins as compared with those of COMTs. Inagreement with this, Hong et al. [23] have also reported thatCOMTs are relatively bigger proteins having molecular weightin the range of 38 to 43 kDa, whereas those of CCoAOMTs aresmaller proteins having molecular weight in the range of 23 to27 kDa.

Domain prediction analysis of the selected COMT se-quences revealed a major methyltransferase 2 superfamilydomain containing AdoMet MTases domain (except for bishop’sweed [Ammi majus]). In addition, they possessed a dimeriza-tion domain at N-terminal. On the other hand, all the analyzedCCoAOMTs possessed a major AdoMet MTases superfamilydomain and a methyltransferase 3 domain. All the COMTsequences analyzed for domain prediction were unique inthe sense that all of them contained a dimerization domain,whereas CCoAOMT sequences lacked this dimerization domain.Presence of a dimerization domain in all the COMTs and itsabsence from all the CCoAOMTs analyzed suggested COMTs asdimers, and CCoAOMTs as monomers, in their native confor-mation, essential for catalysis. Zubieta et al. [5] have reportedthat alfalfa OMT forms a dimer in solution, and this dimericstructure is essential for its activity. On the other hand, Hoff-mann and coworkers [11] have reported that CCoAOMT fromtobacco exists as a catalytically active monomer in solution.Furthermore, alfalfa CCoAOMT has also been shown to cat-alyze transmethylation in monomeric forms [8]. Investigationof localization of COMTs and CCoAOMTs (cytosolic or mem-brane bound) by analyzing transmembrane regions on thesesequences revealed the absence of any transmembrane regionfrom all the sequences, suggesting all of them to be solubleproteins (i.e., cytosolic localization). Similar to our observation,COMTs and CCoAOMTs from wheat, maize, and alfalfa havebeen shown to be localized in cytosolic regions [24,25].

3.2. Conserved motif analysisThe 10 protein sequences from both COMT and CCoAOMTwere analyzed for identifying conserved regions usingClustalW tool. Results are shown in Fig. 1. Thus, among

172 Overlapping Methylation by COMT and CCoAOMT

TABLE 1Isoelectric point (pI), molecular weight, and domain prediction of COMT and CCoAOMT protein sequences from selected plant

sources

COMT CCoAOMT

S. No. Plant source pI Molecular weight (Da) Domainsa pI Molecular weight (Da) Domainsa

1 Ammi majus 6.09 40,151.57 MT 2, DD 5.29 27,042.97 MT 3, AMM

2 Sorghum bicolor 5.46 39,632.54 MT 2, DD 5.23 29,062.21 MT 3, AMM.

3 Zea mays 5.15 39,670.69 MT 2, DD 5.21 29,351.33 MT 3, AMM

4 Oryza sativa 5.23 39,578.49 MT 2, DD 5.21 28,847.77 MT 3, AMM

5 Populous trichocarpa 5.40 39,272.29 MT 2, DD 5.30 27,806.96 MT 3, AMM,

6 Acacia auriculiformis 5.61 39,807.04 MT 2, DD 5.46 28,178.30 MT 3, AMM

7 Leucaena leucocephala 5.60 39,839.16 MT 2, DD 5.31 27,550.63 MT 3, AMM

8 Jatropha curcas 5.52 39,783.18 MT 2, DD 5.29 27,915.89 MT 3, AMM

9 Vitis vinifera 5.48 41,969.34 MT 2, DD 5.27 27,234.28 MT 3, AMM

10 Medicago sativa 5.67 39,946.22 MT 2, DD 5.55 27,999.16 MT 3, AMM

aDomains: MT 2, methyl transferase 2 superfamily; MT 3, methyl transferase 3; DD, dimerization domain; AMM, AdoMet Mtases superfamily.

FIG. 1Conserved regions among COMT (A) andCCoAOMT (B).

the COMT sequences analyzed, seven conserved regions(motifs), namely NEDGVS(143–148), LMNQDK(154–159),LDGGIPFNKAXGMTAFEYHG(174–193), VDVGGGTGA(232–240), GINFDLPHV(254–262), GVEHVGGDMF(272–280), andIMLAHPGGK (348–357), were observed. All of these conservedregions in COMT sequences were found to span between themiddle and C terminus of these sequences. On the basis ofthe multiple sequence alignment of COMT protein sequencesderived from plant sources, Ibrahim et al. [26] have identifiedfive conserved regions (I–V), rich in glycine, toward the mid toC-terminal region of the polypeptide of the protein sequences.Similarly, in our study, the conserved Glycine-rich motifs werealso observed in the mid to C-terminal end of the COMT. In the

case of CCoAOMT, however, nine conserved regions (motifs),namely EVGHKSLL (36–43), LYQYIL (48–53), TSVVPRE (55–61),MTTSADEGQFL (80–90), AKNTMEIGVYTGYSLLATALALP (99–121), EGPALPVLD (156–164), FVDADKDNYLNYH (180–192),GYDNTLWNGSV (206–216), and LPVGDG (253–258), were ob-served. Similarly, Ghosh et al. [27] have also reported eightconserved motifs in Hibiscus cannabinus CCoAOMT, in whichthe three motives are conserved for the SAM binding site. Incomparison with conserved regions of COMT, CCoAOMT se-quences were found to be scattered all along the polypeptidechain. Furthermore, conserved regions in COMTs were foundto be rich in glycine residues, whereas those of CCoAOMTsequences were rich in lysine. It is noteworthy that muchconservation was observed among the various sequences be-longing to COMT and CCoAOMT, separately. However, therewas less conservation between COMT and CCoAOMT.



FIG. 2Dendogram showing phylogenetic relationships ofCOMT (A) and CCoAOMT (B) protein sequencesfrom various plant sources.

3.3. Phylogenetic analysisPhylogenetic analysis of all selected sequences of COMT andCCoAOMT was done using MEGA software. Results are shown inFig. 2. It is noteworthy that these sequences are clustered intotwo separate groups, one group composed of COMT sequencesand the other group composed of CCoAOMT sequences, vali-dating the results of conserved sequence analysis, as describedabove. Furthermore, both COMT and CCoAOMT sequencesbifurcated into two subgroups, namely monocots and dicots.

In the UPGMA-based tree generated with these 20 proteinsequences (10 each of COMT and CCoAOMT), five nodes werestrongly supported as they appeared with a frequency above80%. The three COMTs and CCoAOMTs belonging to monocotswere clearly separated from the other dicots. In the COMTcluster, Vitis vinifera was separated with other dicots with apercentage frequency of 92%; the other branch of this groupis monophyletic, sharing a common ancestor, whereas in thecase of monocot, Oryza sativa was separated with the groupof Zea mays and Sorghum bicolor with a 100% frequency. Inthe CCoAOMT cluster, Acacia magnium was separated withother dicots with a 100% frequency, whereas for other branchforming a monophyletic group, in the dicots group O. sativawas separated with the branch comprising Sorghum bicolorand Zea mays with a 95% frequency.

3.4. 3D structure modeling of COMT and CCoAOMTof M. sativa using MD and DS softwareHomology modeling of COMT and CCoAOMT of M. sativa wasdone using two different software tools, namely MD and DS.For this purpose, COMT (PDB ID 1KYZ) and CCoAOMT (PDB

ID 1SUI) of M. sativa were used as templates. Out of the fivemodels, each of COMT and CCoAOMT generated using twosoftware tools, models of COMT and CCoAOMT with minimumDOPE scores were found to be the most acceptable and henceselected. Both the structures modeled using MD 9v9 and DSversion 3.1 were found to be close. The most acceptable modelof COMT and CCoAOMT as generated using MD 9v9 is shownin Fig. 3. Both these models of COMT and CCoAOMT werefurther validated using PROCHECK, ERRAT, ProSA, and ProQtools. Results are presented in Table 2. The data presented inTable 2 suggest that both the generated models are reliable.

3.5. Superimposition of protein structures of COMTand CCoAOMTModeled COMT and CCoAOMT protein structures were su-perimposed using DS and Chimera. Results are shown inFig. 4. The RMSD score of superimposed structures was found

FIG. 3Modeled structure of COMT (A) and CCoAOMT(B) of M. sativa through Modeller.


TABLE 2Validation of Modeller (MD) and Discovery Studio (DS) generated models of COMT and CCoAOMT of M. sativa using various

tools

Tool Parameter COMT CCoAOMT

MD/ DS Template chosen 1KYZ 1SUI

MD/ DS DOPE score −40,259.19141(MD) −27,489.00977 (MD)

−41,356.4 (DS) −27,277.7 (DS)

PROCHECK (Ramachandran Plot) Percentage of residuesin favored region

93.4% (MD) 88.5% (MD)

92.4% (DS) 89.4% (DS)

ERRAT—overall quality factor Overall quality factora 84.551 (MD) 88.696 (MD)

89.045 (DS) 84.519 (DS)

ProSA Z score −8.91 (MD) −6.94 (MD)

−8.65 (DS) −5.8 (DS)

ProQ Predicted LG score 4.036 (MD) 4.563 (MD)

4.745 (DS) 4.390 (DS)

aOverall quality factor of protein model, expressed as the percentage of the protein for which the calculated error value falls below the 95%rejection limit.

FIG. 4Superimposed structures of COMT (pink) andCCoAOMT (cyan).

to be 21.523, suggesting a poor superimposition of the twostructures. However, at the core of the enzyme structures, asimilarity was observed in spite of the poor sequence similaritybetween the two. In agreement with our results based on the 3D

superimposition of the CCoAOMT and catechol OMT structures,Ferrer et al. [14] reported a high level of similarity in the coreof the enzyme, despite poor sequence similarity.

3.6. Docking studies3.6.1. Comparison of substrate specificities ofCCoAOMTs and COMTsModeled structures of COMT and CCoAOMT of M. sativa weredocked with metabolic intermediates of monolignol biosyn-thesis pathway, namely caffeic acid, sinapic acid, ferulic acid,5-hydroxyferulic acid, their CoA esters, and coniferyl and caf-feoyl alcohols, their aldehydes, 5-hydroxy coniferyl aldehyde,5-hydroxy coniferyl alcohol, SAM, and SAH as putative sub-strates using Autodock as well as CDOCKER and LIBDOCKtools of DS. Results of the docking in terms of binding energyand dissociation constant are presented in Table 3. From thedata presented in Table 3, it is evident that both COMT andCCoAOMT bind all 16 putative substrates efficiently and ef-fectively. The binding energy of various substrates to theseenzymes decreased in the following order: free acid substrates(caffeic acid, sinapic acid, and 5-hydroxyferulic acid) > al-coholic and aldehyde substrates (coniferyl alcohol, ceffeoylalcohol, coniferyl aldehyde, caffeoyl aldehyde, 5-hydroxylconiferyl aldehyde, and 5-hydroxy coniferyl alcohol) > thiolester substrates (caffeoyl-CoA, feruloyl-CoA, 5-hydroxyferuloylCoA, and sinapoyl-CoA), suggesting the best binding for thiolester substrates for both COMT and CCoAOMT. Also on the basisof CDOCKER energy analysis, thiol ester substrates were found



TABLE 3Summary of results of docking of various putative substrates with COMT and CCoAOMT using Autodock

COMT CCoAOMT

Binding energy Dissociation Binding energy DissociationS. No. Substrate (kcal/mol) constant (mM) (kcal/mol) constant (mM)

1 Caffeic acid −3.59 2.32 −5.27 1.03

2 Ferulic acid −3.26 4.07 −5.26 0.138

3 Sinapic acid −4.07 1.04 −4.59 0.430

4 5-Hydroxy ferulic acid −4.06 1.06 −4.44 0.553

5 Coniferyl aldehyde −5.32 0.125 −6.61 0.0142

6 Coniferyl alcohol −5.62 0.075 −7.26 0.0047

7 Caffeoyl aldehyde −5.03 0.205 −6.76 0.011

8 Caffeoyl alcohol −5.38 0.114 −7.37 0.0039

9 5-Hydroxyconiferyl aldehyde −5.39 0.111 −7.36 0.0040

10 5-Hydroxyconiferyl alcohol −5.28 0.134 −6.18 0.29

11 Caffeoyl-CoA −11.92 1.8 × 10−6 −13.86 6.9 × 10−8

12 Feruloyl-CoA −14.24 3.6 × 10−8 −16.38 9.89 × 10−10

13 Sinapoyl-CoA −15.79 2.9 × 10−9 −30.46 4.729 × 10−20

14 5-Hydroxyferuloyl CoA −11.34 4.8 × 10−6 −30.02 9.749 × 10−20

15 S-Adenosyl methionine −4.93 0.243 −5.89 0.048

16 S-Adenosyl homocystine −7.30 0.0044 −7.59 0.0027

to be best for both COMT and CCoAOMT (data not shown).Furthermore, binding energies for all 16 putative substrateswere found to be lower for CCoAOMT than those for COMT,suggesting a better binding of all the substrates to CCoAOMT incomparison with COMT.

Analysis of the dissociation constant (inversely related tothe binding constant of the ligand to the target [enzyme] andthereby with affinity of binding of the ligands to the target) forall the 16 putative substrates to COMT and CCoAOMT revealedthat free acids bind to CCoAOMT with almost 4–10 times higheraffinity than those for COMT (dissociation constant ∼1–4 mMfor COMT and ∼0.1–1.0 mM for CCoAOMT). Similarly, in thecase of aldehydes and alcohols, about 6–25 times higher affin-ity for CCoAOMT than those of COMT (dissociation constant∼0.1–0.2 mM for COMT and ∼0.004–0.03 mM for CCoAOMT)was found. In the case of thiol ester (CoA) derivatives, 10 toseveral thousand times higher affinity for CCoAOMT than thatof COMT (dissociation constant 2.93 × 10−9–4.84 × 10−6 mM forCOMT and 2.18 × 10−20–9.89 × 10−10 mM for CCoAOMT) wasfound. Thus, based on binding energy as well as dissociationconstant, thiol ester derivatives were found to be the bettersubstrates, among all, for CCoAOMT as well as COMT, withsinapoyl-CoA being the best for both enzymes. Furthermore,

between COMT and CCoAOMT, binding with CCoAOMT wasfound to be more effective and efficient than that of COMT.Thus, our in silico studies suggest that alcoholic and aldehy-dic substrates are preferred to those of caffeic, sinapic, andferulic acid by both COMT and CCoAOMT with a marked pref-erence for CoA ester substrates over free acids, aldehydes,and alcohols. More importantly, it is noteworthy that alongwith CCoAOMT, COMT can also methylate CoA esters effi-ciently and is also active toward 5-hydroxy coniferyl alcohol,indicating that COMT can catalyze syringyl unit synthesis inplant and may operate at the free acid, CoA ester, or alcohollevels [4,9].

In agreement with our in silico findings, a number ofreports from in vitro experimental studies are available(Table 4), which suggest broad substrate specificity forboth COMT as well as CCoAOMT, supporting the concept ofmetabolic grid/overlapping pathways in monolignol biosyn-thesis where methylation can occur at various levels andby both of these enzymes. Thus, Zubieta et al. [5] have re-ported that the alfalfa COMT shows marked preference for5-hydroxylated compounds over free acids (Km of 43, 10,and 5 μM for caffeic acid, 5-hydroxyferulic acid, and 5-hydroxyconiferaldehyde, respectively). Parvathi et al. [10]


TABLE 4A comparison of in silico estimated dissociation constants of various putative substrates for COMT and CCoAOMT with the in

vitro experimental Michaelis constant (Km)

In silico estimateddissociation

In vitro experimental data as reported in literature

S. No. Substrate constant (Kd) (mM) Km (mM) Source References

COMT

1 Caffeic acid 2.32 0. 335 Medicago sativa Kota et al. [33]

0.043 Medicago sativa Zubieta et al. [5]

2 5-Hydroxyferulic acid 1.06 0.01 Medicago sativa Zubieta et al. [5]

3 5-Hydroxy coniferyl aldehyde 0.111 0.0018 Medicago sativa Parvathi et al. [10]

4 5-Hydroxyconiferyl alcohol 0.134 0.0075 Medicago sativa Zubieta et al. [5]

0.0038 Medicago sativa Parvathi et al. [10]

0.005 Medicago sativa Zubieta et al. [5]

5 Caffeoyl aldehyde 0.205 0.01 Medicago sativa Zubieta et al. [5]


6 Caffeoyl alcohol 0.114 0.013 Medicago sativa Zubieta et al. [5]


7 S-Adenosyl-L-methionine 0.243 0.012 Medicago sativa Edwards and Dixon [12]

CCoAOMT

1 Caffeic acid 1.03 0.088 Ammi majus Lukacin et al. [30]

0.039 Linumusitatissimum

Day et al. [31]

2 5-Hydroxy-ferulic acid 0.553 0.068 Ammi majus Lukacin et al. [30]

3 Caffeoyl–CoA 6.9 × 10−8 0.0043 Medicago sativa Parvathi et al. [10]

0.020 Ammi majus Lukacin et al. [30]

4 5-Hydroxyferuoyl-CoA 2.1 × 10−20 0.0062 Medicago sativa Parvathi et al. [10]

5 5-Hydroxy-coniferaldehyde 0.0040 0.0173 Triticum estivum Ma and Xu [28]

6 Caffeoyl aldehyde 0.011 0.0437 Triticum estivum Ma and Xu [28]

in alfalfa COMT have also reported COMT-catalyzed methy-lation with a number of potential substrates of monolignolbiosynthesis pathway, with enzyme activities in the follow-ing order: caffeoyl-CoA > caffeoyl alcohol > 5-hydroxyferulicacid > caffeoyl aldehyde > 5-hydroxyconiferyl alcohol > 5-hydroxyferuloyl CoA > 5-hydroxyconiferaldehyde > caffeicacid. Similarly, COMTs from wheat and aspen have also beenshown to catalyze methylation of various free acids, alde-hydic, and alcoholic substrates in the following decreasingorder: 5-hydroxyconiferaldehyde > caffeoyl aldehyde > 5-hydroxyconiferyl alcohol > caffeoyl alcohol > 5-hydroxyferulic

acid > caffeic acid [28, 29]. Maury et al. [9], using recombi-nant COMT and CCoAOMT from tobacco, have studied sub-strate specificity of various metabolic intermediates of phenyl-propanoid pathway, namely caffeic acid, 5-hydroxyferulic acid,their CoA esters, and 5-hydroxyconiferyl alcohol. On the basis ofthe Vmax/Km values, the authors have suggested that both theseenzymes efficiently interacted with CoA ester substrates andcatalyzed the methylation of these substrates more efficientlythan those of free acids. In agreement with these reports, basedon the binding energies and dissociation constants of free acidsand thiol esters substrates, our in silico analyses also revealed



that COMT and CCoAOMT bind CoA ester substrates moreefficiently than those of free acids. CCoAOMTs from bishop’sweed and flax have been reported to methylate free acids andaldehydes along with thiol ester-based substrates such thatmethylation of thiol ester-based substrates was more efficientthan those of free acids and aldehydes [30, 31]. Furthermore,based on the in silico analyses on two isozymes of CCoAOMTfrom Leucaena leucocephala, Pagadala et al. [32] have alsoreported a better binding for caffeoyl-CoA than those of otherCoA substrates for both the isozymes. Thus, our findings are inagreement with the concept of metabolic grid and overlappingpathways in monolignol biosynthesis where methylation canbe done at various levels and by both COMT and CCoAOMT.Thus, based on our in silico data, we have ample evidence topropose that COMT and CCoAOMT may catalyze methylation ofa variety of intermediates of monolignol biosynthesis pathwaywith a comparable efficiency.

3.6.2. Regiospecificity of binding of various putativesubstrates to COMTRegiospecificity of binding of all 16 putative substrates toCOMT, at the active site of enzyme, was investigated andcompared using CDOCKER tool of DS. Results depicting variousinteracting residues are presented in Table 5. Hydrogen bondinteractions between enzyme active-site residues and variousputative substrates along with details of donor/acceptor atomsinvolved in these interactions, their bond lengths, and anglesare summarized in Table 6.

A comparative account of regiospecificity of binding ofvarious substrates at the active site of the COMT in a pairwisemanner involving substrates and products (and vice versa) forrespective reactions is presented below. Caffeic and ferulic acidswere found to interact with a total of 12 and 13 interactingresidues, respectively, out of which, except for MET180 (inthe case of caffeic acid) and LEU127 and ASN324 (in thecase of ferulic acid), all the remaining interacting aminoacid residues of COMT were found to be common. Analysisof hydrogen bonds and other noncovalent interactions amongthese residues of enzymes and various atoms/groups of putativesubstrates revealed that caffeic acid binding involved one eachof hydrogen bond and charge interaction, whereas ferulicacid binding involved only a charge interaction. Thus, HE2 ofHIS269 formed a hydrogen bond with O1 of 3-hydroxyl groupof caffeic acid. HIS323 formed charge interactions with oxygenof carboxylic group of caffeic as well as both ferulic acids.

A comparison of COMT residues, interacting with sinapicacid and 5-hydroxyferulic acid, revealed a total of 12 interactingresidues each; all of these are common. Binding of sinapic acidas well as 5-hydroxyferulic acid both involved one hydrogenbond interaction each. Thus, HE2 of HIS269 formed hydrogenbond interaction with O1 of methoxy group of sinapic acidand O3 of 5-hydroxyl group of 5-hydroxyferulic acid, whereasHIS323 formed charge interactions, one each, with oxygenof carboxyl group of sinapic acid and 5-hydroxyferulic acid,respectively.

Among coniferyl aldehyde and coniferyl alcohol, out of13 interacting residues in each case, 10 interacting residueswere found to be common. TRP266, CYS298, THR356 (in thecase of coniferyl aldehyde) and PHE176, HIS323, ASN324 (inthe case of coniferyl alcohol) were found to be uncommon.Both these substrates involved hydrogen bond interaction withASN131. OD1 of ASN131 formed one hydrogen bond with H23of 4-hydroxyl group of coniferyl aldehydes. On the other hand,HD22 and OD1 of ASN131 formed two hydrogen bonds, onewith O3 and another with H25 of hydroxyl group of propylenechain of coniferyl alcohol, respectively.

In the case of caffeoyl aldehyde and caffeoyl alcohol, out of11 and 13 interacting residues, respectively, 11 residues werefound to be common, except CYS298 and THR356 (present incaffeoyl alcohol). Analysis of hydrogen bonding revealed thatH19 of 3-hydroxyl group of caffeoyl aldehyde involved onehydrogen bond interaction with OD1 of ASN 131, whereas H20and H21 of 3-hydroxyl and 4-hydroxyl group of caffeoyl alcoholwere involved in hydrogen bond interactions with OD1 of ASN131 and SD of MET 130, respectively.

5-Hydroxy coniferyl aldehyde and 5-hydroxy coniferylalcohol each interacted with 13 residues, out of which 12residues were common, whereas ASN131 (in the case of5-hydroxy coniferyl aldehyde) and GLY179 (in the case of 5-hydroxy coniferyl alcohol) were uncommon. HE2 of HIS269formed hydrogen bonds with O3 of the 5-hydroxyl group of5-hydroxy coniferyl aldehyde, whereas HE2 of HIS269 andND1 of HIS183 formed two hydrogen bond interactions withO3 of the 5-hydroxyl group and H24 of the 4-hydroxyl group of5-hydroxy coniferyl alcohol, respectively.

Caffeoyl-CoA and feruloyl-CoA interacted with 31 and34 residues, respectively, out of which 20 residues werefound to be common, whereas THR187, ASP231, LEU232,VAL235, ILE267, TRP271, CYS298, HIS323, PHE354, ASN355,THR356 (in case of caffeoyl-CoA) and LEU32, PRO33, LEU36,ILE89, VAL111, ASN128, GLN132, TRP140, THR211, GLY212,ALA213, VAL214, LYS265, ILE316 (in the case of feruloyl-CoA)were found to be uncommon. Caffeoyl-CoA binding involvedeight hydrogen bond interactions, whereas that of feruloyl-CoAinvolved only two hydrogen bond interactions. HD1 of HIS315,HE2 of HIS183, and O of PHE354 formed three hydrogen bondswith O6, N27, and H96 of adenosyl moiety of caffeoyl-CoA,respectively, whereas HD22 of ASN131 formed two hydrogenbonds with O11 and O14 of the diphosphate moiety of caffeoyl-CoA, respectively. OD1 of ASP231 formed two hydrogen bondswith H94 and H95 of caffeoyl group, whereas OD2 of ASP231formed another hydrogen bond with H95 of caffeoyl groupof caffeoyl-CoA. HD22 of ASN131 and O of GLY209 formedhydrogen bonds with O18 of diphosphate moiety and H98 offeruloyl group of feruloyl-CoA, respectively.

A comparison of sinapoyl-CoA and 5-hydroxyferuloylCoA revealed 27 and 28 interacting residues, respec-tively, out of which 16 residues were found to becommon, whereas MET180, THR187, ASP206, VAL207,GLY209, VAL214, PHE263, MET264, LYS265, CYS298,


TABLE 5Regiospecificity of binding of various putative substrates (ligands) to COMT and CCoAOMT (target) indicating residues involved

in interaction

COMT CCoAOMT(interacting residues and their number in (interacting residues and their number in

Substrate parentheses) in parentheses)

Caffeic acid MET130, ASN131, LEU136, PHE176, MET180,HIS183, TRP266, HIS269, ILE316, ILE319,MET320, HIS323 (12)

GLY19, LYS21, ILE60, ASN190, ASN194,ARG206, ASP238 (7)

Ferulic acid LEU127, MET130, ASN131, LEU136, PHE176,HIS183, TRP266, HIS269, ILE316, ILE319,MET320, HIS323, ASN324 (13)

GLY19, LYS21, SER22, LEU23, ILE60, SER64,ASN194, ASP238 (8)

Sinapic acid LEU127, MET130, ASN131, LEU136, PHE176,HIS183, TRP266, HIS269, ILE316, ILE319,MET320, HIS323 (12)

VAL18, GLY19, LYS21, SER22, LEU23, TYR30,LEU34, ILE60, THR62, SER64, ALA65, ASP66,GLY237, ASP238 (14)

5-Hydroxy ferulicacid

LEU127, MET130, ASN131, LEU136, PHE176,HIS183,TRP266, HIS269, ILE316,ILE319,MET320, HIS323 (12)

VAL18, GLY19, LYS21, SER22, LEU23, TYR30,LEU34, ILE60, THR62, SER64, ALA65, ASP66,GLY237, ASP238 (14)

Coniferyl aldehyde LEU127, MET130, ASN131, LEU136, MET180,HIS183, TRP266, HIS269, CYS298, ILE316,ILE319, MET320, THR356 (13)

VAL18, GLY19, LYS21, SER22, LEU23, TYR30,ILE60, MET61, THR62, THR63, SER64,GLU67, ASP163, ASP189, ASN190, ASN194,GLY237, ASP238 (18)

Coniferyl alcohol LEU127, MET130, ASN131, LEU136, PHE176,MET180, HIS183, HIS269, ILE316, ILE319,MET320, HIS323, ASN324 (13)

VAL18, GLY19, LYS21, SER22, LEU23, ILE60,MET61, THR62, THR63, GLU67, ASP163,ASP189, ASN190, ASN194, GLY237, ASP238(16)

Caffeoyl aldehyde LEU127, MET130, ASN131, GLY179, MET180,HIS183, TRP266, HIS269, ILE316, ILE319,GLU329 (11)

VAL18, GLY19, LYS21, SER22, LEU23, TYR30,ILE60, MET61, THR62, THR63, SER64,GLU67, ASP189, ASN190, GLY237, ASP238(16)

Caffeoyl alcohol LEU127, MET130, ASN131, LEU136, MET180,HIS183, TRP266, HIS269, CYS298, ILE316,ILE319, THR356, GLU329 (13)

GLY19, HIS20, LYS21, SER22, ILE60, MET61,THR62, THR63, SER64, GLU67, ASP189,ASN190, GLY237, ASP238 (14)

5-Hydroxy coniferylaldehyde

LEU127, MET130, ASN131, LEU136, PHE176,MET180, HIS183, TRP266, HIS269, ILE316,ILE319, MET320, HIS323 (13)

VAL18, GLY19, LYS21, SER22, LEU23, TYR30,ILE60, THR62, SER64, ASN190, ASN194,GLY237, ASP238 (13)

5-Hydroxy coniferylalcohol

LEU127, MET130, LEU136, PHE176, GLY179,MET180, HIS183, TRP266, HIS269, ILE316,ILE319, MET320, HIS323 (13)

LYS21, SER22, LEU23, TYR30, ILE60, MET61,THR62, THR63, SER64, ASP66, ASP189,ASN194, GLY237, ASP238 (14)

Caffeoyl-CoA ILE124, LEU127, MET130, ASN131, LEU136,PHE163, PHE176, MET180, HIS183, THR187,GLY208, GLY210, ASP231, LEU232, VAL235,TRP266, ILE267, HIS269, ASP270, TRP271,CYS298, GLY312, HIS315, ILE316, ILE319,MET320, HIS323, ASN324, PHE354, ASN355,THR356 (31)

SER12, GLY13, ARG14, HIS15, GLN16, GLU17,VAL18, GLY19, HIS20, LYS21, SER22, LEU23,ASP27, ALA28, TYR30, GLN31, LEU34,PHE39, ILE60, MET61, THR62, THR63,SER64, ALA65, ASP66, GLY87, VAL88,TYR89, ASP111, ILE112, ASP163, ALA164,ASP165, LYS166, ASN190, TRP193, ASN194,TYR208, TYR212, GLU237, ASP238 (41)

(Continued)



TABLE 5Continued

COMT CCoAOMT(interacting residues and their number in (interacting residues and their number in

Substrate parentheses) in parentheses)

Feruloyl-CoA LEU32, PRO33, LEU36, ILE89, VAL111, ILE124,LEU127, ASN128, MET130, ASN131,GLN132, LEU136, TRP140, PHE163, PHE176,MET180, HIS183, ASP206, GLY208, GLY210,THR211, GLY212, ALA213, VAL214, LYS265,TRP266, HIS269, ASP270, GLY312, HIS315,ILE316, ILE319, MET320, ASN324 (34)

GLU11, SER12, GLY13, ARG14, HIS15, GLN16,GLU17, VAL18, GLY19, HIS20, LYS21, SER22,LEU23, ASP27, ALA28, TYR30, GLN31,LEU34, PHE39, ILE60, MET61, THR62, THR63,SER64, ALA65, ASP66, GLY87, VAL88,TYR89, ASP111, ILE112, ASP163, ALA164,ASP165, LYS166, ASN190, TRP193, ASN194,TYR208, TYR212, GLY237, ASP238 (42)

Sinapoyl-CoA MET130, ASN131, PHE163, PHE176, MET180,HIS183, THR187, ASP206, VAL207, GLY209,VAL214, PHE263, LYS265, MET264, ILE267,HIS269, TRP266, ASP270, CYS298, LYS311,GLY312, HIS315, ILE316, ILE319, MET320,HIS323, ASN324 (27)

GLU11, SER12, GLY13, HIS15, GLN16, GLU17,VAL18, GLY19, HIS20, LYS21, SER22, LEU23,ASP27, ALA28, TYR30, GLN31, LEU34,PHE39, TRP58, ILE60, MET61, THR62, THR63,SER64, ALA65, GLY87, VAL88, TYR89,ASP111, ASP163, ALA164, ASP165, LYS166,ASN190, TRP193, ASN194, TYR208, TYR212,GLY237, ASP238 (40)

5-HydroxyferuloylCoA

LEU36, VAL111, TYR114, LEU115, LEU127,ASN128, MET130, ASN131, ALA162,PHE163, HIS166, PHE176, HIS183, GLY208,GLY210, ASP231, ILE267, TRP266, HIS269,ASP270, TRP272, GLY312, HIS315, ILE316,ILE319, MET320, HIS323, ASN324 (28)

SER12, GLY13, HIS15, GLN16, GLU17, VAL18,GLY19, HIS20, LYS21, SER22, ASP27, ALA28,TYR30, GLN31, LEU34, PHE39, ILE60,MET61, THR62, SER64, ALA65, ILE160,ASP163, LYS166, ASP189, ASN190, TRP193,ASN194, ARG206, TYR208, VAL209, TYR212,GLN237, ASP238 (34)

S-Adenosylmethionine

LEU127, MET130, ASN131, LEU136, ALA162,PHE163, HIS166, PHE176, MET180, HIS183,TRP266, HIS269, GLU297, CYS298, ILE316,ILE319, MET320, HIS323, GLU329, THR356(20)

VAL18, GLY19,HIS20, LYS21, SER22, LEU23,TYR30, LEU34, ASN59, ILE60, MET61,THR62, THR63, SER64, GLU67, ASP163,ASP189, ASN190, ASN194, ASP238 (20)

S-Adenosylhomocystine

LEU127, MET130, ASN131, LEU136, ALA162,PHE163, PHE176, MET180, HIS183, TRP266,HIS269, ASP270, GLY312, VAL313, HIS315,ILE316, ILE319, MET320, HIS323, ASN324(20)

VAL18, GLY19, LYS21, SER22, LEU23, TYR30,LEU34, ILE60, MET61, THR62, THR63,SER64, ALA65, GLU67, ASP189, ASN190,ASN194, GLY237, ASP238 (19)

and LYS311 (in the case of sinapoyl-CoA) and LEU36, VAL111,TYR114, LEU115, LEU127, ASN128, ALA162, HIS166, GLY208,GLY210, ASP231, and TRP272 (in the case of 5-hydroxyferuloylCoA) were found to be uncommon. Sinapoyl-CoA binding to theenzyme involved a total of eight hydrogen bond interactions.Thus, HD 1 of HIS315 formed a hydrogen bond with N26 ofadenosyl moiety, HD22 of ASN131 formed a hydrogen bondwith O15 of diphosphate moiety, HE2 of HIS269 formed twohydrogen bonds with O16 and O19 of pantothenate moiety,HG of CYS298 formed two hydrogen bonds with O9 and H100of 3′phosphate of ribose moiety, HD21 of ASN355, HE2 ofHIS183 formed hydrogen bonds with O9 and O12, respec-tively, of 3′phosphate of ribose moiety of sinapoyl-CoA. On the

other hand, 5-hydroxyferuloyl CoA binding involved only threehydrogen bond interactions, HD22 of ASN128 formed a hydro-gen bond with O21 of feruloyl moiety, HE2 of HIS269 formedtwo hydrogen bonds with O9 and O12 of 3′phosphate of ri-bose moiety and one charge interaction with the same moiety,whereas HIS323 formed charge interaction with oxygen ofdiphosphate moiety of 5-hydroxyferuloyl CoA.

In the case of SAM (methyl group donor) and SAH, out of 20interacting residues in each case 15 residues were found to becommon, whereas HIS166, GLU297, CYS298, GLU329, THR356(in case of SAM) and ASP270, GLY312, VAL313, HIS315,ASN324 (in case of SAH) were uncommon. SAM binding to theenzyme involved a total of seven hydrogen bond interactions.


TAB

LE

6H

yd

rog

en

bo

nd

inte

racti

on

sb

etw

een

acti

ve-s

ite

resid

ues

of

CO

MT

an

dC

Co

AO

MT

an

dvari

ou

sp

uta

tive

su

bstr

ate

salo

ng

wit

h

deta

ils

of

do

no

r/accep

tor

ato

ms

invo

lved

inth

ese

inte

racti

on

san

dth

eir

bo

nd

len

gth

san

dan

gle

sas

decip

here

du

sin

gC

DO

CK

ER

CO

MT

CC

oAO

MT

Dis

tanc

eD

ista

nce

betw

een

dono

rbe

twee

ndo

nor

Acc

epto

ran

dac

cept

orB

ond

Acc

epto

ran

dac

cept

orB

ond

S.N

o.S

ubst

rate

Don

orat

omat

omat

om(A

)an

gle

(◦ )D

onor

atom

atom

atom

(A)

angl

e(◦ )

1C

affe

icac

id(C

A)

HIS

269:

HE

2C

A:O

12.

0303

914

4.07

1LY

S21

:HZ

2C

A:O

32.

2824

913

9.03

8

––

––

LYS

21:H

Z2

CA

:O4

2.27

868

113.

555

2Fe

rulic

acid

(FA

)–

––

–LY

S21

:HZ

2FA

:O4

1.89

828

156.

613

3S

inap

icac

id(S

A)

HIS

269:

HE

2S

A:O

12.

2226

713

2.24

3A

RG

50:H

ES

A:O

42.

2688

616

0.50

4

––

––

SA

:H27

LYS

21:O

2.07

945

143.

365

45-

Hyd

roxy

feru

licac

id(5

HFA

)H

IS26

9:H

E2

5HFA

:O3

2.36

885

130.

712

AR

G50

:HE

5HFA

:O5

2.43

575

159.

591

––

––

5HFA

:H23

LYS

21:O

2.34

3212

1.36

5

5C

on

ifer

ylal

deh

yde

(Co

Ald

)C

oA

ld:H

23A

SN

131:

OD

12.

0178

149.

279

SE

R22

:HG

Co

Ald

:O2

2.18

246

143.

392

––

––

AS

N19

0:H

D22

Co

Ald

:O3

2.44

772

108.

55

6C

on

ifer

ylal

coh

ol

(Co

Alc

)A

SN

131:

HD

22C

oA

lc:O

32.

0910

614

2.78

5T

HR

63:H

G1

Co

Alc

:O3

2.44

379

140.

592

Co

Alc

:H25

AS

N13

1:O

D1

2.01

225

146.

635

Co

Alc

:H24

LYS

21:O

2.03

808

160.

386

––

––

Co

Alc

:H25

ME

T61

:O1.

9487

716

6.05

2

7C

affe

oyl

ald

ehyd

e(C

aAld

)C

aAld

:H19

AS

N13

1:O

D1

1.94

807

157.

429

SE

R22

:HG

CaA

ld:O

22.

2517

613

3.01

6

––

––

AS

N19

0:H

D22

CaA

ld:O

32.

0620

415

2.54

7

––

––

CaA

ld:H

23G

LY19

:O2.

0698

814

1.93

2

––

––

CaA

ld:H

19G

LY19

:O2.

0118

314

6.57

3

8C

affe

oyl

alco

ho

l(C

aAlc

)C

aAlc

:H20

AS

N13

1:O

D1

1.97

487

150.

126

AS

N19

0:H

D22

CaA

lc:O

12.

3631

916

1.46

2

CaA

lc:H

21M

ET

130:

SD

2.37

689

139.

039

CaA

lc:H

20M

ET

61:O

2.04

222

170.

332

––

––

CaA

lc:H

21A

SP

189:

OD

22.

1638

112

7.90

9

––

––

CaA

lc:H

22LY

S21

:O1.

9952

413

7.26

1

(Con

tinue

d)



TAB

LE

6C

on

tin

ued

CO

MT

CC

oAO

MT

Dis

tanc

eD

ista

nce

betw

een

dono

rbe

twee

ndo

nor

Acc

epto

ran

dac

cept

orB

ond

Acc

epto

ran

dac

cept

orB

ond

S.N

o.S

ubst

rate

Don

orat

omat

omat

om(A

)an

gle

(◦ )D

onor

atom

atom

atom

(A)

angl

e(◦ )

95-

Hyd

roxy

con

ifer

ylal

deh

yde

(5H

Co

Ald

)H

IS26

9:H

E2

5HC

oA

ld:O

31.

9935

132.

418

SE

R22

:HG

5HC

oA

ld:O

32.

1671

214

1.87

––

––

5HC

oA

ld:H

23G

LY19

:O2.

4258

213

0.33

9

105-

Hyd

roxy

con

ifer

ylal

coh

ol(

5HC

oA

lc)

HIS

269:

HE

25H

Co

Alc

:O3

2.26

441

111.

405

SE

R22

:HG

5HC

oA

lc:O

41.

8270

414

7.62

1

––

––

AS

N19

0:H

D22

5HC

oA

lc:O

12.

1123

715

4.73

7

5HC

oA

lc:H

24H

IS18

3:N

D1

2.43

104

131.

578

5HC

oA

lc:H

26G

LY23

7:O

2.41

951

152.

972

11C

affe

oyl

-Co

A(C

Co

A)

AS

N13

1:H

D22

CC

oA

:O11

2.46

727

127.

404

AS

N19

0:H

D21

CC

oA

:O18

2.27

188

121.

956

AS

N13

1:H

D22

CC

oA

:O14

2.04

916

139.

031

CC

oA

:H91

TY

R21

2:O

H2.

3584

210

4.85

6

HIS

183:

HE

2C

Co

A:N

272.

2602

814

0.59

9C

Co

A:H

99G

LY19

:O1.

9384

114

9.64

2

HIS

315:

HD

1C

Co

A:O

62.

0697

415

2.63

3–

––

–

CC

oA

:H94

AS

P23

1:O

D1

2.19

745

147.

815

––

––

CC

oA

:H95

AS

P23

1:O

D1

2.06

679

145.

028

––

––

CC

oA

:H95

AS

P23

1:O

D2

2.02

796

136.

368

––

––

CC

oA

:H96

PH

E35

4:O

2.32

5111

0.37

5–

––

–

12Fe

rulo

yl-C

oA

(FC

oA

)A

SN

131:

HD

22FC

oA

:O18

2.25

115

120.

051

GLY

13:H

NFC

oA

:O23

2.44

148

121.

878

FCo

A:H

98G

LY20

9:O

1.96

725

129.

722

LYS

166:

HZ

1FC

oA

:O7

1.79

045

144.

483

––

––

AS

N19

0:H

D22

FCo

A:O

82.

2756

411

1.93

5

––

––

AS

N19

0:H

D22

FCo

A:O

172.

1868

213

0.03

3

––

––

FCo

A:H

97LY

S21

:O1.

9344

915

2.15

––

––

FCo

A:H

100

AS

P11

1:O

D1

2.46

7717

6.96

6

––

––

FCo

A:H

100

AS

P11

1:O

D2

2.15

927

123.

127

––

––

FCo

A:H

102

TY

R30

:OH

2.11

178

125.

813

(Con

tinue

d)


TAB

LE

6C

on

tin

ued

CO

MT

CC

oAO

MT

Dis

tanc

eD

ista

nce

betw

een

dono

rbe

twee

ndo

nor

Acc

epto

ran

dac

cept

orB

ond

Acc

epto

ran

dac

cept

orB

ond

S.N

o.S

ubst

rate

Don

orat

omat

omat

om(A

)an

gle

(◦ )D

onor

atom

atom

atom

(A)

angl

e(◦ )

13S

inap

oyl

-Co

A(S

Co

A)

AS

N13

1:H

D22

SC

oA

:O15

2.42

749

124.

597

ALA

65:H

NS

Co

A:O

202.

3890

415

5.40

7

HIS

183:

HE

2S

Co

A:O

121.

8855

215

7.39

LYS

166:

HZ

1S

Co

A:O

52.

2541

713

1.02

2

HIS

269:

HE

2S

Co

A:O

162.

1426

213

8.40

1A

SN

190:

HD

22S

Co

A:O

112.

1432

614

4.73

2

HIS

269:

HE

2S

Co

A:O

192.

1949

413

1.90

5A

SN

190:

HD

22S

Co

A:O

182.

2799

112

3.07

CY

S29

8:H

GS

Co

A:O

92.

2983

97.2

247

SC

oA

:H69

ME

T61

:SD

2.33

597

139.

905

HIS

315:

HD

1S

Co

A:N

262.

2483

514

7.46

9S

Co

A:H

72LY

S21

:O1.

9713

213

0.63

8

AS

N35

5:H

D21

SC

oA

:O9

2.00

787

142.

985

SC

oA

:H73

ILE

60:O

1.86

732

143.

623

SC

oA

:H10

0C

YS

298:

SG

2.40

703

138.

429

––

––

145-

Hyd

roxy

feru

loyl

Co

A(5

HFC

oA

)A

SN

128:

HD

225H

FCo

A:O

212.

4595

411

2.06

2LY

S16

6:H

Z1

5HFC

oA

:O14

2.49

223

87.9

172

HIS

269:

HE

25H

FCo

A:O

92.

1154

513

4.21

7LY

S16

6:H

Z3

5HFC

oA

:O14

1.99

856

118.

879

HIS

269:

HE

25H

FCo

A:O

122.

2159

212

1.10

1A

SP

189:

HD

25H

FCo

A:O

171.

8724

512

1.21

5

––

––

AS

N19

0:H

D22

5HFC

oA

:O13

1.98

778

167.

183

––

––

5HFC

oA

:H97

ME

T61

:O2.

2748

813

0.91

7

––

––

5HFC

oA

:H10

2IL

E60

:O2.

4614

313

0.48

15S

-Ad

eno

syl

met

hio

nin

e(S

AM

)T

RP

266:

HE

1S

AM

:O5

2.32

881

121.

371

ALA

65:H

NS

AM

:O6

2.20

271

169.

461

TR

P26

6:H

E1

SA

M:O

62.

1458

312

2.29

2A

SN

190:

HD

22S

AM

:N8

2.29

782

132.

763

CY

S29

8:H

NS

AM

:O6

2.34

915

102.

213

SA

M:H

46T

YR

30:O

H2.

1083

616

9.65

3

SA

M:H

45A

SN

131:

OD

12.

2314

215

4.08

2S

AM

:H47

GLY

19:O

2.00

229

135.

148

SA

M:H

46G

LU32

9:O

E1

2.29

379

134.

092

SA

M:H

50A

SP

189:

OD

22.

4498

211

9.99

6

SA

M:H

47G

LU29

7:O

E1

1.85

212

139.

175

––

––

SA

M:H

48G

LU32

9:O

E2

1.95

962

128.

671

––

––

(Con

tinue

d)



TAB

LE

6C

on

tin

ued

CO

MT

CC

oAO

MT

Dis

tanc

eD

ista

nce

betw

een

dono

rbe

twee

ndo

nor

Acc

epto

ran

dac

cept

orB

ond

Acc

epto

ran

dac

cept

orB

ond

S.N

o.S

ubst

rate

Don

orat

omat

omat

om(A

)an

gle

(◦ )D

onor

atom

atom

atom

(A)

angl

e(◦ )

16S

-Ad

eno

syl

ho

mo

cyst

ein

e(S

AH

)H

IS26

9:H

E2

SA

H:O

42.

2724

812

4.05

4A

SP

238:

HN

SA

H:O

52.

2401

712

4.18

6

HIS

315:

HD

1S

AH

:O5

2.19

237

151.

429

SA

H:H

43T

YR

30:O

H1.

9549

716

6.20

5

SA

H:H

44G

LY31

2:O

1.79

083

134.

25S

AH

:H45

TH

R62

:O1.

8322

113

6.75

3

––

––

SA

H:H

46M

ET

61:O

1.87

344

124.

169

Thus, H45 of the adenosyl moiety formed a hydrogen bond withOD1 of ASN131, HE1 of TRP266 formed hydrogen bonds withthe O5 and O6 of the methionine moiety, H46 and H48 of theamino group formed a hydrogen bond with OE1 and OE2 ofGLU329, respectively, whereas H47 of the amino group formeda hydrogen bond with OE1 of GLU297. HN of CYS298 formeda hydrogen bond with O6 of methionine moiety and HIS269formed charged interactions with sulfur atom and nitrogen ofthe amino group. GLU297 and GLU329 also formed chargedinteractions with the nitrogen of the amino group. TRP266formed π–cation interaction with the sulfur atom, whereasHIS323 formed π–π interaction with the two rings of adeninemoiety. HIS269 formed π–cation interaction with the nitrogenof NH3 group. SAH binding involved only three hydrogen bondinteractions. Thus, HE2 of HIS269 formed a hydrogen bondwith the O4 of the adenosyl moiety, whereas HD1 of HIS315with O5 of methionine moiety. H44 of the amino group formeda hydrogen bond with O of GLY312.

Among all the substrates analyzed, interacting residuesMET130, HIS183, HIS269, ILE316, and ILE319 were found tobe common. Interacting residues for our best docked substrate,among all analyzed substrates, namely sinapoyl-CoA withCOMT, as a representative case are shown in Fig. 5.

On the basis of a crystallographic study of alfalfa COMT,Zubieta et al. [5] have reported that the hydroxyl group of hy-drophobic residues MET130, PHE176, MET180, and MET320interacted with SAM. Furthermore, the hydrophobic bindingpocket containing LEU136, PHE172, PHE176, and ALA162was also reported to interact well with methoxy groups ofboth ferulic acid and 5-hydroxyconiferaldehyde, suggestingthat these van der Waal interactions were responsible forthe kinetic preferences of COMT toward the 3-methoxy-4,5-dihydroxyl-substituted substrates over the 3,4-dihydroxyl-substituted substrates. The authors have also reported thatthe residues HIS183, ASN131, MET180, MET130, ILE316, andILE319 were interacted with the propanoid tail of substratesand this relative hydrophobicity of the binding pocket is sug-gestive of the selectivity for neutral aldehydes and alcoholsover negatively charged carboxylate groups. Their findingsare in agreement with our docking analysis data where theresidues that were involved in the binding (interaction) offree acids, alcohols, aldehydes, SAM, and SAH are commonand similar to what Zubieta et al. [5] have reported. Thus,the residues that were common between our analysis andthose reported by Zubieta et al. [5] are MET130, ASN131,LEU136, PHE176, MET180, HIS183, TRP266, HIS269, ILE316,ILE319, MET320, and HIS323. Furthermore, Gordon et al.[34], based on the site-directed mutagenesis of ryegrass COMT,have reported that HIS plays a crucial role in COMT catalysisas the substitution of HIS resulted in the complete loss of en-zyme activity. From our study also, HIS looks to be a significantresidue as it is common in binding/interaction of all the putativesubstrates.


FIG. 5Two-dimensional (2D) view of the dockedsinapoyl-CoA with COMT showing interactingresidues involved in sinapoyl-CoA binding.

3.6.3. Regiospecificity of binding of various putativesubstrates to CCoAOMTRegiospecificity of binding of all 16 putative substrates toCCoAOMT, at the active site of the enzyme, was investigatedand compared using CDOCKER tool of DS. Results are presentedin Table 5. Hydrogen bond interactions between enzyme active-site residues and various putative substrates along with detailsof donor/acceptor atoms involved in these interactions, theirbond lengths, and angles are summarized in Table 6.

Data on regiospecificity of binding of various putativesubstrates to CCoAOMT revealed that caffeic and ferulic acidsbinding to CCoAOMT involved a total of seven and eightresidues, respectively, out of which all the residues, except forASN190, ARG206 (in case of caffeic acid) and SER22, LEU23,SER64 (in case of ferulic acid), were common. Caffeic acidbinding involved two hydrogen bonds, whereas that of ferulicacid involved one hydrogen bond. Thus, HZ2 of LYS21 formedtwo hydrogen bonds with O3 and O4 of the carboxyl groupof caffeic acid; in addition, LYS21 and ARG206 also formedcharged interactions with the oxygen of the carboxylic group ofcaffeic acid. In the case of ferulic acid, HZ2 of LYS21 formed ahydrogen bond with the O4 of the carboxyl group and a chargedinteraction with oxygen of the carboxyl group of ferulic acid.

A comparison of interacting residues for sinapic acidand 5-hydroxyferulic acid with CCoAOMT revealed that all14 interacting residues were common. Both these substratesinvolved two H-bond interactions each, involving LYS21 andARG50. Thus, H27 and O4 of the 4-hydroxyl group of sinapic

acid were involved in the hydrogen bond interaction, oneeach with O of LYS21 and HE of ARG50, respectively, whereasin the case of 5-hydroxyferulic acid O of LYS21 and HE ofARG50 formed hydrogen bonds, one each with the H23 of the4-hydroxyl group and O5 of 5-hydroxyferulic acid, respectively.

Among coniferyl aldehyde and coniferyl alcohol out of18 and 16 interacting residues, respectively, 16 interactingresidues were found to be common, with TYR30 and SER64being unique to coniferyl aldehyde. Coniferyl aldehyde bindinginvolved two hydrogen bond interactions, whereas that ofconiferyl alcohol involved three hydrogen bond interactions.Thus, HG of SER22 formed a hydrogen bond with O2 of the 4-hydroxyl group, whereas HD22 of ASN190 formed a hydrogenbond with the O3 of the carboxyl group of coniferyl aldehyde.H24 of the 4-hydroxyl group, H25 and O3 of the hydroxyl groupof propylene chain of coniferyl alcohol formed hydrogen bondsone each, with O of LYS21, O of MET61, and HG1 of THR63,respectively.

In the case of caffeoyl aldehyde and caffeoyl alcohol, 16and 14 interacting residues, respectively, were observed, outof which 13 residues were common, whereas VAL18, LEU23,TYR30 (in the case of caffeoyl aldehyde), and HIS20 (in thecase of caffeoyl alcohol) were uncommon residues. Caffeoylaldehyde and caffeoyl alcohol both involved four hydrogenbond interactions each. Thus, HG of SER22 formed a hydrogenbond with O2 of the 4-hydroxyl group, whereas O of GLY19formed two hydrogen bonds with H23 and H19 of the 3-hydroxylgroup and HD22 of ASN190 formed a hydrogen bond with theO3 of the aldehydic group of caffeoyl aldehyde, respectively.In caffeoyl alcohol, H20 and H21 of 3- and 4-hydroxyl groupsformed hydrogen bonds with O of MET61 and OD2 of ASP189,respectively, whereas HD22 of ASN190 formed one hydrogen



bond with the O1 of the 3-hydroxyl group and H22 of thehydroxyl group of propylene chain of caffeoyl alcohol formed ahydrogen bond with O of LYS21.

Interacting residues in the case of 5-hydroxy coniferylaldehyde and 5-hydroxy coniferyl alcohol were 13 and 14,respectively, out of which 10 residues were common withVAL18, GLY19, ASN190 (in the case of 5-hydroxy coniferylaldehyde) and THR62, THR63, ASP66, ASP189 (in the caseof 5-hydroxy coniferyl alcohol) were uncommon. 5-Hydroxyconiferyl aldehyde involved two hydrogen bond interactions,whereas 5-hydroxy coniferyl alcohol involved three hydrogenbond interactions. Thus, HG of SER22 and O of GLY19 formedone hydrogen bond each with O3 of the 5-hydroxyl group andH23 of the 4-hydroxyl group of 5-hydroxy coniferyl aldehyde,respectively. In case of 5-hydroxy coniferyl alcohol, HD22 ofASN190 and HG of SER22 formed a hydrogen bond, one each,with the O1 of the 3-methoxy group and O4 of the hydroxylgroup of propyl chain of 5-hydroxy coniferyl alcohol, whereasH26 of the alcoholic group formed a hydrogen bond with O ofGLY237.

In the case of caffeoyl-CoA and feruloyl-CoA, there were atotal of 41 and 42 interacting residues, respectively, out of which41 interacting residues were common, with GLU11 (in feruloyl-CoA) as uncommon. Binding of caffeoyl-CoA to the enzymeinvolved three hydrogen bond interactions, whereas that offeruloyl-CoA involved eight hydrogen bond interactions. Thus,in caffeoyl-CoA, H99 of the NH group of mercaptoethylamine ofCoA moiety formed a hydrogen bond with O of GLY19. HD21 ofASN190 formed a hydrogen bond with O18 of the diphosphatemoiety of caffeoyl-CoA, H91 of adenosyl moiety of caffeoyl-CoAformed a hydrogen bond with OH of TYR212. LYS166 formedthe π–cation interaction with the adenine ring of CoA. In thecase of feruloyl-CoA, H97 of OH group of mercaptoethylamineof CoA moiety formed a hydrogen bond with O of LYS21, HN ofGLY13 formed a hydrogen bond with O23 of 4-hydroxyl group offeruloyl moiety, HD22 of ASN190 formed hydrogen bonds, oneeach, with the O8 and O17 of the diphosphate moiety, HZ1 ofLYS166 formed a hydrogen bond with the O7 of the adenosylmoiety, H100 of the amino group of adenosyl moiety formed twohydrogen bonds, one each, with OD1 and OD2 of ASP111. H102of NH group of mercaptoethylamine of CoA moiety formed ahydrogen bond with OH of TYR 30. ASP238 formed a chargedinteraction with oxygen of the diphosphate group of CoA moiety.

Comparing sinapoyl-CoA and 5-hydroxyferuloyl CoA re-vealed 40 and 34 interacting residues, respectively, out ofwhich 30 residues were common with GLU11, LEU23, TRP58,THR63, GLY87, VAL88, TYR89, ASP111, ALA164, ASP165 (inthe case of sinapoyl-CoA) and ILE60, ASP189, ARG206, VAL209(in the case 5-hydroxyferuloyl-CoA) as uncommon residues.Sinapoyl-CoA binding involved seven hydrogen bond inter-actions, whereas that of 5-hydroxyferuloyl CoA involved sixhydrogen bond interactions. Thus, in sinapoyl-CoA, H69 of the3′phosphate group of phosphoadenosine diphosphate moietyformed a hydrogen bond with SD of MET61, H73 of the OHgroup of diphosphate moiety formed a hydrogen bond with O

of ILE60, whereas H72 of the OH group of pentothenate moietyformed a hydrogen bond with O of LYS21, HN of ALA65 formeda hydrogen bond with O20 of β-mercaptoethylamine moiety,HD22 of ASN190 formed two hydrogen bonds, one each, withO11 and O18 of diphosphate moiety and HZ1 of LYS166 formeda hydrogen bond with O5 of adenosyl moiety of sinapoyl-CoA.In 5-hydroxyferuloyl CoA, H97 of OH group, and H102 of NHgroup of pantothenate moiety formed one hydrogen bond eachwith O of MET61 and O of ILE60, HD2 of ASP189, and HD22 ofASN190 formed one hydrogen bond each with O17 and O13 ofdiphosphate moiety, HZ1 and HZ3 of LYS166 formed two hydro-gen bonds with O14 of diphosphate moiety of 5-hydroxyferuloylCoA, respectively. ASP163 and LYS166 formed four chargedinteraction, two each, with oxygen of diphosphate moiety.

A comparison of SAM and SAH binding involved 20 and 19interacting residues, respectively, out of which 17 interactingresidues were common, whereas HIS20, ASN59, ASP163 (inSAM) and ALA65, GLY237 (in SAH) were uncommon. SAMbinding involved five hydrogen bond interactions, whereasthat of SAH involved four hydrogen bond interactions. Thus,in SAM, H46, H47 of the amino group of methionine moietyformed hydrogen bonds one each, with OH of TYR30 and Oof GLY19, respectively. H50 of the amino group of adenosylmoiety formed a hydrogen bond with OD2 of ASP189, HN ofALA65, and HD22 of ASN190 formed a hydrogen bond withN8 of the adenine moiety. ASP238 formed π–sigma interactionwith the adenine moiety of SAM. In the case of SAH, H43 of theamino group of adenine moiety formed a hydrogen bond withOH of TYR30, whereas H45 and H46 of the amino group ofhomocysteine moiety formed hydrogen bonds with O of THR62and O of MET61, respectively. ASP238 formed a hydrogenbond with O5 of homocysteine moiety and was also involved incharge interaction with oxygen of homocysteine moiety.

A comparison of interacting residues of CCoAOMT with allthe 16 putative substrates revealed that GLY19, LYS21, SER22,LEU23, ILE60, ASN190, ASN194, and ASP238 are the residuesthat were common for almost all the substrates. Interactingresidues for our best docked substrate among all analyzed,namely, sinapoyl-CoA with CCoAOMT as a representative case,are shown in Fig. 6.

Our in silico findings regarding residues involved in inter-action with these substrates, are in agreement with those re-ported from the in vitro experimental studies. Thus, Ferrer et al.[14], while studying the interaction of various CoA substrates,namely, feruloyl-CoA, sinapoyl-CoA, and 5-hydroxyferuloylCoA in the crystal structures of alfalfa CCoAOMT, have re-ported that residues LYS21, MET61, ASP163, ASN190, TRP193,ARG206, TYR208, and TYR212 are involved in the formation ofa common substrate-binding pocket. In our in silico analyses,these residues are found to be involved in the formation of asimilar common substrate-binding pocket at the active site ofCCoAOMT. Similarly, Hoffmann et al. [13], working with tobaccoCCoAOMT, have reported involvement of an ASN residue, whichis conserved in all CCoAOMTs, having a crucial role in CCoAOMTcatalysis. Furthermore, the authors, by doing site-directed


FIG. 62D view of the docked sinapoyl-CoA withCCoAOMT showing interacting residues involvedin sinapoyl-CoA binding.

mutagenesis, have shown that substitution of ARG220 witha THR resulted in the total loss of enzyme activity, whereaschanges of ASP58 to ALA and GLN61 to SER were shownto decrease affinity toward CoA, thereby suggesting that theARG220 might be involved in the electrostatic interaction withthe coenzyme A moiety of the thiol ester substrate [13]. Alsoin the present in silico study, ARG, ASP, and GLN residueshave been found to be common for almost all CoA substrates,suggesting their crucial roles. On the basis of the in silico anal-yses on isozymes of CCoAOMT from Leucaena leucocephala,Pagadala et al. [32] have reported the involvement of MET,THR, VAL, GLU, GLY, SER, ASP, THR, ASN, and ARG residues atthe active site of the isozymes, which are suggested to maintainthe positive charge at the active sites for the efficient binding ofthe most favored substrate, that is, negatively charged caffeoyl-CoA. A similar observation is also evident from our in silicoanalyses. Results of the regiospecificity of binding of varioussubstrates to the enzymes and analysis of noncovalent interac-tions (especially hydrogen bonds) suggest that binding of thepair of putative substrates (substrates/products) is mediated bychange in the conformation in the enzymes (both COMT andCCoAOMT). A similar observation on enzyme conformationalchanges in binding of substrates and products has also beensuggested in the case of Leucaena leucocephala CCoAOMT [32].

3.6.4. Comparison of regiospecificity of bindingof various putative substrates between COMTand CCoAOMTA comparison of regiospecificity of binding of various putativesubstrates to COMT and CCoAOMT revealed that the majorityof residues involved in binding of these substrates (interactingresidues) at the active site of COMT and CCoAOMT are common,suggesting a similar mode of binding of these substrates to their

enzymes (Table 5). Thus, a comparison of interacting residuesbetween COMT and CCoAOMT revealed that ILE is presentin all the substrates. LEU is present in 13 substrates; it isnot common only in the case of caffeic acid, caffeoyl alcohol,and sinapoyl-CoA. ASN is common in 13 substrates; it is notpresent in sinapic acid, 5-hydroxy ferulic acid, and 5-hydroxyconiferyl aldehyde. MET is common in 11 substrates; it is notcommon in all the free acids and 5-hydroxy coniferyl aldehyde.HIS is common in six substrates; it is not present in all thefree acids, alcohols, aldehydes, and SAH. THR is commononly in five substrates, that is, coniferyl aldehyde, caffeoylalcohol, SAM, caffeoyl-CoA, and sinapoyl-CoA. GLY is present ineight substrates, namely 5-hydroxy coniferyl alcohol, caffeoylalcohol, SAH, and all the CoA esters. ASP is common in all theCoA esters and in SAH. TRP, PHE, and VAL are common in onlythe CoA esters. GLU is common in three substrates, caffeoylalcohol, caffeoyl adehyde, and SAM. LYS is common only inferuloyl-CoA and sinapoyl-CoA. GLN is common only in thecase of feruloyl-CoA. Thus, overall it can be concluded that thebinding cavity of both COMT and CCoAOMT has 14 commonresidues, namely ILE, LEU, ASN, MET, HIS, THR, GLY, ASP, TRP,PHE, VAL, GLU, LYS, and GLN.

Data based on in vitro wet laboratory studies have shownthat the SAM binding sites of various O-methyltransferases areconserved. Thus, the methionine part of SAM was reported tointeract with THR and LYS, whereas that of the ribose part ofSAM with ASP in the case of rice COMT [5,13,35]. Similarly, inour study THR was also found to interact with the methioninepart of SAM in the case of COMT, whereas that of CCoAOMTinteracted with both THR and LYS, and the ribose part of SAMinteracted with ASP in the case of CCoAOMT, whereas no suchinteraction was found in the case of COMT.

A comparison of conformation of binding of various sub-strates to COMT and CCoAOMT revealed that the majority ofsubstrates bound to COMT and CCoAOMT in a similar con-formation except for the thiol esters (CoA-based substrates).Thus, a comparison of the data presented in Figs. 5 and 6



clearly indicated that sinapoyl-CoA binds to COMT in a dif-ferent conformation than that of CCoAOMT. On the basis ofthe energy and affinity constant values, it may be suggestedthat binding of sinapoyl-CoA to CCoAOMT is more favoredthan that of COMT. The significance of these data on variousconformations of substrate binding to the enzymes is difficult toexplain at present but a comparison of data on Km and Vmax ofCOMT and CCoAOMT for these substrates from in vivo/in vitrowet laboratory studies suggests that both enzyme–substratescomplexes (COMT+ sinapoyl-CoA and CCoAOMT+ sinapoyl-CoA) are productive [9]. Therefore, it may be inferred that thesubstrates that are so far not considered as the conventionalsubstrates of these enzymes may also bind these enzymes in anefficient manner and are catalytically active even if they bindin a different conformation to enzymes. In some cases, thesechanged conformations are more effective.

4. ConclusionsLignin, which makes up an average 25% of the plant biomass,is considered to be one of the greatest obstacles to the optimalutilization of the plant biomass for various purposes such aspaper manufacturing, production of highly palatable forage,and bagasse utilization. Therefore, there has been a worldwideeffort in the past two decades for the reduction/alternation oflignin in plant biomass using molecular genetic approaches,for their better utilization. In view of alternative methylationpathways reported for more than a decade, it has been sug-gested that COMTs as well as CCoAOMTs both can efficientlycatalyze the methylation of substrates that are traditionallynot considered as their normal substrates. However, there isstill ambiguity with regard to substrate affinities and efficiencyof catalysis for COMTs and CCoAOMTs. Therefore, this needsfurther validation. In this direction, the present work dealswith analysis and comparison of 10 protein sequences, forboth COMT and CCoAOMT, from selected dicots and monocotsfor parameters like molecular weights, pI, domain analysis,their cellular localization, the evolutionary relationship, andsubstrate specificity. All the COMT and CCoAOMT sequencesanalyzed were found to be acidic and soluble in nature.CCoAOMTs were found to be smaller proteins of monomericnature as compared with those of COMTs, which were dimericwith a unique dimerization domain. Phylogenetic analysis ofthe COMT and CCoAOMT sequences revealed their clusteringinto two major groups: one group composed of COMT, and theother group composed of CCoAOMT; these two groups werefurther subgrouped into monocots and dicots. A comparisonof the modeled COMT and CCoAOMT of M. sativa revealeda similarity at the core region of the protein with a uniquedomain in COMT, which might be involved in the dimerization.The docking analysis of COMT and CCoAOMT with the 16putative substrates (intermediates of monolignol biosynthe-sis pathway) revealed that all these substrates bind to bothCOMT and CCoAOMT, with binding to CCoAOMT being moreefficient. Results of the present molecular docking analyses

have suggested newer roles of these enzymes in methylation ofintermediates of monolignol biosynthesis pathway.

5. AcknowledgementsFinancial support from the Department of Biotechnology,Govt. of India, New Delhi, under the BIF programme andDepartment of Higher Education, Govt. of U.P., under theCenter of Excellence programme is gratefully acknowledged.

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