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Molecular docking and dynamics simulation analysesunraveling the differential enzymatic catalysis by plantand fungal laccases with respect to lignin biosynthesisand degradationManika Awasthia, Nivedita Jaiswala, Swati Singha, Veda P. Pandeya & Upendra N. Dwivediaa Bioinformatics Infrastructure Facility, Center of Excellence in Bioinformatics, Departmentof Biochemistry, University of Lucknow, Lucknow 226007, IndiaAccepted author version posted online: 10 Oct 2014.Published online: 06 Nov 2014.

To cite this article: Manika Awasthi, Nivedita Jaiswal, Swati Singh, Veda P. Pandey & Upendra N. Dwivedi (2014):Molecular docking and dynamics simulation analyses unraveling the differential enzymatic catalysis by plant and fungallaccases with respect to lignin biosynthesis and degradation, Journal of Biomolecular Structure and Dynamics, DOI:10.1080/07391102.2014.975282

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Molecular docking and dynamics simulation analyses unraveling the differential enzymaticcatalysis by plant and fungal laccases with respect to lignin biosynthesis and degradation

Manika Awasthi, Nivedita Jaiswal, Swati Singh, Veda P. Pandey and Upendra N. Dwivedi*

Bioinformatics Infrastructure Facility, Center of Excellence in Bioinformatics, Department of Biochemistry, University of Lucknow,Lucknow 226007, India

Communicated by Ramaswamy H. Sarma

(Received 28 August 2014; accepted 7 October 2014)

Laccase, widely distributed in bacteria, fungi, and plants, catalyzes the oxidation of wide range of compounds. Withregards to one of the important physiological functions, plant laccases are considered to catalyze lignin biosynthesiswhile fungal laccases are considered for lignin degradation. The present study was undertaken to explain this dual func-tion of laccases using in-silico molecular docking and dynamics simulation approaches. Modeling and superimpositionanalyses of one each representative of plant and fungal laccases, namely, Populus trichocarpa and Trametes versicolor,respectively, revealed low level of similarity in the folding of two laccases at 3D levels. Docking analyses revealed sig-nificantly higher binding efficiency for lignin model compounds, in proportion to their size, for fungal laccase as com-pared to that of plant laccase. Residues interacting with the model compounds at the respective enzyme active sites werefound to be in conformity with their role in lignin biosynthesis and degradation. Molecular dynamics simulation analysesfor the stability of docked complexes of plant and fungal laccases with lignin model compounds revealed that tetramericlignin model compound remains attached to the active site of fungal laccase throughout the simulation period, while itprotrudes outwards from the active site of plant laccase. Stability of these complexes was further analyzed on the basisof binding energy which revealed significantly higher stability of fungal laccase with tetrameric compound than that ofplant. The overall data suggested a situation favorable for the degradation of lignin polymer by fungal laccase while itssynthesis by plant laccase.

Keywords: homology modeling; laccase; lignin biosynthesis and degradation; molecular docking; molecular dynamicssimulation

1. Introduction

Laccases (benzenediol: oxygen oxidoreductase; EC1.10.3.2) are multicopper oxidases, widely distributed inbacteria, fungi, higher plants, and insects (Hoegger,Kilaru, James, Thacker, & Kues, 2006). They belong toblue copper oxidase superfamily and have the ability tocatalyze oxidation of a wide range of compounds such assubstituted phenols, diamines, aromatic amines, thiols,and even some inorganic compounds. The ability of lac-cases, to catalyze the oxidation of a range of compounds,makes them more versatile with regards to their functionas well as industrial applications. Thus, the diverse func-tions mediated by laccases include polymerization/depoly-merization of lignin, fungal pathogenesis, wound healing,sclerotization, morphogenesis, sporulation, pigmentation,fruiting body formation, melanin formation, endosporecoat protein synthesis etc. (Dwivedi, Singh, Pandey, &Kumar, 2011). Among these diverse functions of laccases,the lignification/delignification has been considered as oneof the very significant function because of their involve-ment in various industrial applications such as pulp and

paper manufacturing, biobleaching, bioenergy production,biomass conversion, biofuel, and removal of a largenumber of environmental pollutants, such as alkenes,chlorophenols, dyes, herbicides, polycyclic aromatichydrocarbons, and benzopyrene. (Gianfreda, Xu, &Bollag, 1999; Jaiswal, Pandey, & Dwivedi, 2014; Lange,& Grell, 2014; Rodríguez Couto, Herrera, & Toca, 2006).

In plants, the lignification process encompasses thepolymerization of monolignols via their dehydrogenationfollowed by combinatorial free-radical coupling by acouple of enzymes including laccases in the cell wall.Based on experimental studies, it has been reported thatlaccases from several plant species efficiently oxidizemonolignols to dehydrogenative polymers (Bao, O’malley,Whetten, & Sederoff, 1993; Chabanet et al., 1994; Davin,Bedgar, Katayama, & Lewis, 1992; Driouich, Laine, Vian,& Faye, 1992; Liu, Dean, Friedman, & Eriksson, 1994;McDougall, & Morrison lan, 1996; Sterjiades, Dean, &Eriksson, 1992). Expression of laccase, predominantly inthe secondary xylem, have been reported from trees likePopulus trichocarpa (Ranocha et al., 1999) and Pinus

*Corresponding author. Email: dwivedi_un@lkouniv.ac.in

© 2014 Taylor & Francis

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taeda (Sato, Wuli, Sederoff, & Whetten, 2001), suggest-ing its role in lignin biosynthesis in plants. Evidence forparticipation of laccase in lignin biosynthesis has alsobeen reported on the basis of plants transformationstudies using laccase gene constructs (Berthet et al.,2011; Ranocha et al., 2002).

In fungi, on the other hand, lignin biodegradation,also an oxidative process, involves laccase mediatedbreakdown of lignin polymer and oxidative degradationof the side chains of the p-hydroxyphenyl (H), guaiacyl(G), and syringyl (S) lignin units, releasing a set ofphenolic compounds (acids, ketones, and aldehydes)(Camarero, Galletti, & Martinez, 1994). The variousproducts of these oxidative degradation reactions havealso been reported to act like mediators (endogenous)and thereby, further promote the oxidative degradation ofthe lignin polymer (Camarero, Ibarra, Martinez, &Martinez, 2005; Cañas et al., 2007; Eggert, Temp, Dean,& Eriksson, 1996; Nousiainen, Maijala, Hatakka,Martínez, & Holzforschung, 2009; Torres-Duarte,Roman, Tinoco, & Vazquez-Duhalt, 2009). The degrada-tion of the lignin model compounds namely, syringylglycol-β-guaiacyl ether and syringyl type β-aryl ether bylaccase from Polyporus versicolor and Stereum frustula-tum have also been demonstrated (Kirk, Harkin, &Cowling, 1968; Wariishi, Morohoshi, & Haraguchi,1987). Moreover, a diminished lignin degrading abilityin laccase-minus mutants of Sporotrichzum puluerulen-tum while enhanced lignolytic activity in laccase-plusmutants have been reported (Ander & Eriksson, 1976).

The fundamental molecular architecture of laccasecopper center, distributed into three redox sites namelyT1, T2, and T3 sites, at the active site of laccases, isquite similar in both plant and fungal laccases (Claus,2004). Despite this similarity in molecular architecture,plant and fungal laccases have been reported to possesswide phylogenetic, physicochemical, as well as func-tional diversity (Colaneri & Vitali, 2014; Dwivedi et al.,2011; Satpathy, Behera, Padhi, & Guru, 2013).

Thus, in the light of the reports stated above, it isevident that plant laccases are involved in lignin biosyn-thesis while fungal laccases in lignin degradation ordepolymerization. However, the basis of the dual func-tion of laccases catalyzing lignin biosynthesis (plants)and degradation (fungi) has not yet been elucidatedunequivocally. Thus, few reports have shown that redoxpotential of laccase play a crucial role in deciding thefate of the reaction, i.e. whether lignification or delignifi-cation. Fungal laccases due to their higher redox poten-tial (up to + 800 mV) are capable of acting on both thephenolic and non-phenolic subunits of lignin thereby,playing a role in lignin degradation. Plant laccases poly-merize lignin by coupling of the phenoxy radicals due toits lower redox potential (430 mV) (Li, Xu, & Eriksson,1999). This difference in the redox potential of laccases

has been reported due to the involvement of differentamino acid residues at the T1 copper site. Plant laccaseshave a methionine residue in addition to one cysteineand two histidine residues to construct the T1 copper site(Nitta, Kataoka, & Sakurai, 2002), while in fungal lac-cases phenylalanine is found to play the role (Morozova,Shumakovich, Gorbacheva, Shleev, & Yaropolov, 2007).Xu et al. (1998) observed that Trametes versicolor lac-case, having high redox potential (800 mV), have aphenylalanine residue instead of methionine and pre-dicted that it might be responsible for the high redoxpotential. Methionine forms a strong axial bond to T1copper, while phenyalanine do not bind to copper andthus increase hydrophobicity surrounding T1 copper.Due to absence of bonds to the axial ligand, the Cu–Scysbond is much stronger which accounts for the increase inredox potential of fungal laccases. pH dependences offungal and plant laccases has also been suggested as oneof the basis for this dual action of laccases in lignin deg-radation or synthesis (Gorbacheva et al., 2008; Madhavi& Lele, 2009). Thus, fungal laccases exhibit low pHoptima which may be due to their adaptation to growunder acidic conditions, while plant laccases being intra-cellular have their pH optima nearer to the physiologicalrange. Thus, the differences in pH optima might be sug-gested to be linked to this dual function of laccases.

In addition, the overall three-dimensional structure oflaccase, leading to altered microenvironment at the activesite of the enzyme, has also been suggested as the basisof the dual action of lignin biosynthesis and degradationcatalyzed by the two laccases. Thus, based on the crys-tallographic studies of two distinct types of fungal lac-cases, one involved in lignin degradation (T. versicolor[basidiomycetes]) while other involved in lignin biosyn-thesis (Melanocarpus albomyces [ascomycetes]) (similarto that of plant laccase), Hakulinen et al. (2002) havereported that in the laccase of T. versicolor, oxygenenters in the tri-nuclear cluster through an open tunnel,whereas in laccase of M. albomyces, the C-terminusforms a movable plug which can block this access andtrap the oxygen. This C-terminal blockage of theM. albomyces laccases significantly reduces the speed ofthe free inflow of O2 and release of water moleculesthereby, making the surrounding environment moreappropriate for polymerization while, in case ofT. versicolor laccases, the rapid exchange of O2 andwater does not allow for the build-up of free radicals inthe microenvironment, and thus avoiding polymerization.Thus, the authors have proposed that the architecturaldifferences at the C-terminal end of M. albomyces andT. versicolor laccases might be responsible for their rolein lignification and delignification, respectively.

A comparison of some of the salient features of plantand fungal laccases, illustrating broader differencesamong them, are presented in Table 1.

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The present work was initiated with the objectiveof providing a newer basis of the yet unequivocallyexplained dual role of lignin biosynthesis and degrada-tion exhibited by plant and fungal laccases, using rele-vant bioinformatical tools, which is of both academicas well as of practical interest looking to the wideapplication of laccases. Thus, laccases from two repre-sentative classes belonging to each of plant and fungihave been analyzed at the levels of protein sequence,3D structure, as well as molecular dynamics interac-tions using relevant in silico tools. For copper bindingmotif analyses, 30 protein sequences each of the twoclasses of laccase (plant and fungal) were retrieved andanalyzed. Three-dimensional structures of P. trichocarpaand T. versicolor laccase, representatives of each plantand fungal laccase, respectively, were modeled anddocked with various lignin model compounds of differ-ent complexity levels, to compare the active site inter-actions in both the representative classes of laccases.The docked complexes were further subjected to 25 nsmolecular dynamics simulations to investigate and com-pare the binding efficiency and stability of variouscompounds with the modeled laccases. Results of thein-silico analyses were validated and discussed in lightof data available from experimental studies.

2. Materials and methods

2.1. Sequence analysis

Thirty homologous laccase protein sequences, each fromplant and fungi, were selected from NCBI database(http://www.ncbi.nlm.nih.gov). The multiple sequence

alignment (MSA) for each of the plant and fungal groupof laccase sequences were performed by COBALT MSAtool on default parameters (Papadopoulos & Agarwala,2007). Copper binding motif analyses of the laccasesequences were carried out using ScanProsite tool(http://prosite.expasy.org/scanprosite).

2.2. Homology modeling of plant and fungal laccases

For modeling, a plant laccase protein sequence from P.trichocarpa while that of from a fungi T. versicolor, asrespective representative classes, were selected. In orderto select a template for homology modeling of laccases,BLASTP (Altschul, Gish, Miller, Myers, & Lipman,1990) analyses were performed. Based on high similarityscore, crystal structure of ascorbate oxidase (1AOZ) andlaccase complexed with 2,5-xylidine (1KYA) were usedas template structures for modeling of plant and fungallaccases, respectively. Protein sequences of both the lac-cases and their corresponding templates were alignedaccurately and 3D models were generated using ‘BuildHomology Models’ module of Discovery Studio 3.5(DS) (Accelrys Software Inc., 2013). The ‘Loop Refine-ment’ and ‘Side-Chain Refinement’ modules were usedfor refinement of initial 3D models of laccase. Finally,the energy minimizations were performed usingCHARMM27 forcefield by 500-step steepest-descentminimization followed by conjugate gradient minimiza-tion. The final laccase models were further validatedusing ‘Modeler’ module of DS for protein verificationand tested for stereochemical accuracy with the‘Procheck’ online program on SAVES server (http://nih

Table 1. A comparison of the salient features of plant and fungal laccases.

Properties Plant laccases Fungal laccases

A) Biochemical1) pH optima range 7.0–10.0 (neutral to alkaline)a,b 2.0–6.5 (acidic)c,d

2) Isoelectric point (pI) 5.0–9.0e 3.0–5.0f

3) Molecular weight On an average > 100 kDaa,b On an average <100 kDaa

4) Glycosylation High: ~45%e,f Low: 10–25%e,f

5) Redox potential Low (400–500 mV)g High (700–800 mV)h

B) Functions Lignin biosynthesis, wound healing, flavonoidoxidation, and cell wall formationf

Lignin degradation, pathogenesis, detoxification,fungal development, and morphogenesisf

C) Significant active siteresidues for catalysis

MET, ASNi PHE, ASPi

D) Evolutionaryrelationship

Form widely separated phylogenetic clustersj Form widely separated phylogenetic clustersj

aJaiswal, Pandey, and Dwivedi (2014).bJaiswal, Pandey, and Dwivedi (2015).cLi, Zhang, Wang, and Ng (2010).dNicolini, Bruzzese, Cambria, Bragazzi, and Pechkova (2013).eBerthet et al. (2012).fSolomon et al. (2014).gLi, Xu, and Eriksson (1999).hXu et al. (1998).iXu (1996).jDwivedi et al. (2011).

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server.mbi.ucla.edu/SAVES/). Residue compatibility wasassessed by the ‘Profile-3D’ module in DS. Modeledplant and fungal laccases were further superimposed oneach other using ‘Align and Superimpose Proteins’module of DS.

2.3. Preparation of lignin model compounds

Lignin model compounds namely, sinapyl alcohol(monomer), guaiacyl 4-O-5 guaiacyl (dimer), syringyl β-O-4 syringyl β-O-4 sinapyl alcohol (trimer), and guaiacylβ-O-4 syringyl β-β syringyl β-O-4 guaiacyl (tetramer)were selected from NMR database of lignin and cell wallmodel compounds (Ralph, Ralph, & Landucci, 2004),and their structures were sketched (Table 3). Energy min-imization was performed using CHARMm forcefield andconformations were generated for each compound usingBEST algorithm.

2.4. Molecular docking of laccases with various ligninmodel compounds

The active sites of the selected representative classes ofplant and fungal laccases were predicted based on thePDB site records using ‘Define and Edit Binding Sitemodule’ and subsequently docking of the selected ligninmodel compounds was performed using ‘LibDock mod-ule’ of DS. Docking interactions between laccases andselected lignin model compounds were analyzed with thehelp of the ‘Analyze complexes’ module of DS. Basedon the docking score, the best conformation of eachcompound was selected for further analyses.

2.5. Molecular dynamics simulations of dockedcomplexes

Stability of the docked complexes and interactions ofplant and fungal laccases with the selected lignin modelcompounds were investigated through 25 ns of moleculardynamics (MD) simulations using GROMACS 4.5.5package (Pronk et al., 2013) with GROMOS96 43a1force field. The docking poses of laccase with respectivelignin model compounds were prepared for MD simula-tion through mild minimization and solvation within awater filled 3-D cube of 1 Å spacing using simple pointcharge (SPC), a three-point model for water. A leap-frogtime integration algorithm was used for integrating New-ton’s equations of motion. System was neutralized andfurther minimized. The complex structure was heated to300 K and equilibrated for 100 ps in NVT ensemble andanother 100 ps in NPT ensemble. After heating andequilibration, the laccase–lignin model compound com-plex was subjected to production run of 25 ns in NPTensemble. PRODRG web server (SchuÈttelkopf & vanAalten, 2004) was used to generate topologies and

coordinates of ligands. Default values of GROMACSwere assigned for determination of hydrogen bondinginteractions and interaction energies between laccase andlignin model compounds.

2.6. Binding free-energy analyses

The binding free energies of the complexes between lac-cases and various lignin model compounds, during theMD simulation analyses, were computed usingg_mmpbsa tool of GROMACS (Kumari, Kumar, OSDDConsortium, & Lynn, 2014) based on the molecularmechanics/Poisson–Boltzman surface area (MM/PBSA)method (Kollman et al., 2000). The binding energy cal-culations were performed for 1000 snapshots taken at aninterval of 10 ps during the 15–25 ns (equilibrium phase)of each trajectory of MD simulation.

3. Results and discussion

3.1. Sequence alignment analysis

MSA for 30 each of plant and fungal laccases weredone. An in depth analysis of MSA, results revealed thepresence of several conserved stretches of 20–30 aminoacid residues. The position of these stretches in align-ment lies in range of 158–178 and 649–669 for fungiand 170–190 and 658–678 for plants. These twostretches of 21 amino acid residues each show highestdegree of conservation, i.e. ≥90% within the group and≥70% with other group, as compared to other stretches.To explore the functional role of these conservedstretches, a search was made against PROSITE database,which revealed the match of these conserved stretcheswith multicopper oxidase pattern (PS00079 andPS00080) of PROSITE. The pattern consisted of four H-X-H motifs (copper binding region) which form copperinteraction site with highly conserved histidine residues(Supplementary Figures S1 and S2). Valderrama, Oliver,Medrano-Soto, and Vazquez-Duhalt (2003) have reportedthat the highly conserved histidine residues interact withcopper ion during laccase-mediated catalysis. The promi-nent conservation at multicopper domains amonglaccases has been suggested to play key role inlaccase-mediated catalysis as well as in its evolution(Messerschmidt & Huber, 1990; Solomon, Sundaram, &Machonkin, 1996).

Comparative analysis of H-X-H motif shows aremarkable difference at position X within group as wellas outside the group. In the first H-X-H motif, trypto-phan was found to be highly conserved in both plantsand basidiomycetes, however, in ascomycetes a phenylal-anine was found. The second H-X-H motif shows higherconservation within group but more variation out ofgroup. In basidiomycetes, serine shows higher conserva-tion while ascomycetes and plants are characterized by

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alanine. The third and fourth H-X-H motif showed high-est conservation within the group as well as outside thegroup as compared to first two motifs explained above.The X residue of third motif (position between T2 andT3) represents leucine in plants and basidiomycetes,while lysine in ascomycetes. The fourth motif, character-ized by HCH, showed 100% conservation within as wellas outside the groups. These observed differences maybe significant from the point of view of enzyme catalysiswith regards to lignin biosynthesis/degradation.

3.2. 3D structure modeling of plant and fungallaccases

The homology modeling generated five models for eachof selected plant and fungal laccases with differentDOPE scores. The respective models with minimumDOPE scores (Figure 1) were selected for further analy-ses. The modeled laccases with minimum DOPE scoreswere further validated using Modeler, Procheck, and Pro-file-3D, and were found to be reliable for interpretationof structure–function relationships (Table 2). The analy-ses of 3D structures of the two laccases revealed a simi-lar folding having three sequentially arrangedcupredoxin-like domains. These domains are formed byβ-sheets and β-strands, arranged in sandwich conforma-tion (Lindley, 2001). The results were in agreement withthose reported for similar representative laccases fromX-ray cryatallographic analyses and literature survey(Dwivedi et al., 2011; Kallio et al., 2011; Piontek,Antorini, & Choinowski, 2002).

Comparison of the modeled plant and fungal laccasesin the present study revealed prominent differences in

the folding of C-terminal region. The role of C-terminalresidues of fungal laccases in enzyme catalysis and sta-bility has been investigated in detail by Bleve et al.(2013) by performing various deletions as well as substi-tutions using site-directed mutagenesis. The C-terminalregion of plant laccase formed an extended structurewhich could easily form a movable plug, as has beenreported for M. albomyces (Hakulinen et al., 2002),where it has been suggested to help in trapping of theoxygen during laccase-mediated lignin biosynthesis. Onthe other hand, in the case of fungal laccase, the C-ter-minal part of the protein formed a coiled structure,imparting rigidity in the movement and thereby allowingfree flow of oxygen. In agreement to our findings, Haku-linen et al. (2002) have also reported a distinct differenceat the C-terminal end in the crystal structure of M. alb-omyces and T. versicolor laccase, and suggested that thisdifference may be the reason for the dual behavior oflaccase. Thus, in the present study, plant laccase wasfound to have similar 3D structure as proposed for M.albomyces (Hakulinen et al., 2002). Since, plant laccasepossessed similar function to M. albomyces laccase, i.e.polymerization of monolignols, therefore, the mechanismof free radical generation through C-terminal blockage ofO2 could also be applied to plant laccase. Moreover, M.albomyces laccase also exhibited low redox potentialsimilar to plant laccase.

3.2.1. Superimposition analysis of modeled laccases

Superimposition analysis of modeled plant and fungallaccases revealed a root-mean-square deviation (RMSD)of 7.25 Å with 454 overlapping residues (Figure 2)

Figure 1. Three-dimensional structures of plant (A) and fungal (B) laccases generated by homology modeling.

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suggesting a not-so-significant similarity at 3D structurelevel among these laccases. Furthermore, pairwisesequence alignment of primary sequences of the twomodeled laccases revealed 27.6% sequence identity and45.9% sequence similarity with highly conserved copperbinding motifs.

3.3. Docking studies of modeled laccases with ligninmodel compounds

Molecular docking of both plant and fungal laccaseswith various lignin model compounds was done usingDS. Results are shown in Table 3. Results of docking

analyses revealed that binding efficiency (based on dock-ing scores) of lignin model compounds increased withthe size of the model compounds in both fungal as wellas plant laccases; however, the degree of the increase inbinding efficiency was found to be significantly higherfor fungal laccase as compared to that of plant laccase.Thus, the ratio of LibDock scores (LDS) of fungal lac-case to that of plant laccase were found to be 1.419 formonomer, 1.457 for dimer, 1.556 for trimer, and 2.037for tetramer corresponding to a fold increase of 41.91%for monomer, 45.73% for dimer, 55.64% for trimer, and103.71% for tetramer (Table 3). Thus, based on the pres-ent study, it could be concluded that larger compoundssuch as trimers and tetramers interact much more effi-ciently with the fungal laccase than that of plant laccasesuggesting a possible reason for dual activity exhibitedby these laccases.

3.3.1. Regiospecificity of binding of various ligninmodel compounds to plant and fungal laccases

Regiospecificity of binding of various lignin model com-pounds at the active site of plant and fungal laccaseswas compared. Results depicting various interacting resi-dues are presented in Table 3. Plant laccase was foundto interact with a total of 11, 16, 23, and 21 amino acidresidues in the case of monomer, dimer, trimer, and tetra-mer, respectively, out of which, 8 residues namelyMET287, ASP288, ASN333, PRO454, GLU455,SER456, THR500, and HIS525 were found to be com-mon in the binding of all the model compounds. Fungallaccase was found to interact with a larger number ofamino acid residues, i.e. 14, 17, 23, and 23 in the caseof monomer, dimer, trimer, and tetramer, respectively,out of which, 11 residues namely LEU185, ASP227,ASN229, PHE260, SER285, PHE286, GLY413,ALA414, PRO415, ILE476, and HIS479 were found tobe common in the binding of all the model compounds.In agreement with our results, Kallio et al. (2009) whileelucidating the crystal structure of M. albomyces laccaseco-crystallized with a lignin model compound haveshown involvement of seven residues, namely ALA,PRO, GLU, LEU, PHE, TRP, and HIS in the binding of

Table 2. Validation of modeled structure of plant and fungal laccases using various tools.

Tools Parameter Plant laccase Fungal laccase

Modeler DOPE score −57, 394.4 −60, 562Procheck (Ramachandran plot) Most favored regions 88.1% 90.2%

Additional allowed regions 10.8% 9.3%Generously allowed regions .2% .5%Disallowed regions .9% .0%

Profile-3D Verify score 189.03 217.92Verify expected high score 244.115 227.991Verify expected low score 109.852 102.596

Figure 2. 3D superimposition of modeled structures of plant(green) and fungal (red) laccases.

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Table 3. Docking results of plant and fungal laccases with lignin model compounds.

S.No. Names Structure

LibDock score(LDS) Ratio of

LDS(Fungal:plant)

Increase offungal LDS overthat of plant

(%)*

Interacting residues

Plantlaccase

Fungallaccase Plant laccase

Fungallaccase

1 Sinapyl alcohol(monomer)

63.997 90.8197 1.419 41.91 GLU230,MET287,

LEU185,ASP227,

ASP288,ASN333,

PRO228,ASN229,

ALA453,PRO454,

PHE260,ALA261,

GLU455,SER456,

SER285,PHE286,

THR500,LEU522,

GLY413,ALA414,

HIS525 PRO415,PRO417,ILE476,HIS479

2 Guaiacyl 4-O-5guaiacyl (dimer)

65.8983 96.0372 1.457 45.73 THR181,ASN228,

PHE183,LEU185,

GLU230,MET287,

ASP227,PRO228,

ASP288,ASN333,

ASN229,GLN258,

PRO454,GLU455,

PHE260,ALA261,

SER456,HIS457,

SER285,PHE286,

PRO458,THR500,

GLY413,ALA414,

LEU522,VAL524,

PRO415,HIS416,

HIS525,TRP528

PRO417,ILE476,HIS479

3 Syringyl β-O-4syringyl β-O-4 sinapylalcohol (trimer)

75.0253 116.768 1.556 55.64 TRP131,TRP167,

PHE183,PRO184,

THR181,GLY182,

LEU185,GLY186,

LEU183,LEU227,

ASP227,ASN229,

ASN228,PHE286,

HIS230,THR231,

MET287,ASP288,THR289,PHE330,

GLN258,PHE260,

MET331,ASN333,

ASN283,PRO284,

ILE451,ALA453,

SER285,PHE286,

PRO454,GLU455,

VAL289,PRO412,

SER456,THR500,

GLY413,ALA414,

VAL524,HIS525,

PRO415,THR451,

TRP528 ILE476,PHE478,HIS479

(Continued)

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lignin model compound at the active site of laccase. Thesame set of residues was found to interact with the ligninmodel compounds in the present study. Histidine resi-dues have been shown to interact strongly with aromaticrings in a size dependent manner, through π–π stackinginteractions with aromatic rings (such as lignin modelcompounds, used in present study), leading to catalysis(Rocha, Ramalho, Caetano, & da Cunha, 2013).

With regards to dual function of lignin biosynthesisand degradation exhibited by plant and fungal laccases,respectively, in the present study, the fungal laccaseswhich have high redox potential was found to involvephenylalanine in the binding of all the lignin model com-pounds, while in case of plant laccases which are charac-terized by a low redox potential, a methionine was foundto be involved in binding of these model compounds. Inagreement to the present analyses, Morozova et al.(2007) have reported that the high-potential laccaseshave a phenylalanine residue as an axial ligand of the T1site copper while in low-potential laccases this role isplayed by methionine residue. Based on site-directedmutagenesis, Xu (1996) has also reported that laccases,harboring PHE at Type I copper site, exhibited highredox potential, whereas laccases with MET exhibitedlow redox potential. It has also been suggested that ASPresidue at the binding site of fungal laccase plays a cru-cial role in proton abstraction from the substrate, but in

plant laccase ASN residue instead of ASP, performs thesame role (Frasconi, Favero, Boer, Koivula, & Mazzei,2010; Madzak et al., 2006). Similar to these reports, inour study, an ASP227 has been observed at the activesite of fungal laccase, while that of ASN228 in plant lac-case. Furthermore, the binding of monomer, trimer, andtetramer to plant and fungal laccases exhibited conforma-tional changes also (Supplementary Figure S3), therebysuggesting the basis for differences in LibDock scoresand this might be the possible reason for the dual cata-lytic differences in plant and fungal laccases.

3.4. Molecular dynamics simulation analysis

For MD simulation studies, two types of lignin modelcompounds at the two extremes, namely a monomer (si-napyl alcohol) and a tetramer (guaiacyl β-O-4 syringylβ-β syringyl β-O-4 guaiacyl), were selected. Thus, a totalof four 25 ns MD simulations, two each for monomerand tetramer with plant and fungal laccases, were done.Results for RMSD for plant and fungal laccases com-plexed with monomeric and tetrameric lignin modelcompounds are shown in Figure 3. It is noteworthy thatboth plant and fungal laccases complexed with mono-meric/tetrameric lignin model compounds maintainedequilibrium between 15 and 25 ns of simulation. All fur-ther analyses were performed during this equilibrium

Table 3. (Continued).

S.No. Names Structure

LibDock score(LDS) Ratio of

LDS(Fungal:plant)

Increase offungal LDS overthat of plant

(%)*

Interacting residues

Plantlaccase

Fungallaccase Plant laccase

Fungallaccase

4 Guaiacyl β-O-4syringyl β-β syringylβ-O-4 guaiacyl(tetramer)

80.0528 163.074 2.037 103.71 THR181,GLY182,

PHE183,LEU185,

LEU183,ASN228,

ASP227,ASN229,

MET287,ASP288,

THR231,GLN258,

THR289,VAL291,

PHE260,ASN283,

PHE330,MET331,

PRO284,SER285,

ASP332,ASN333,

PHE286,VAL289,

ILE451,ALA453,

GLN314,PHE353,

PRO454,GLU455,

GLY355,PHE358,

SER456,THR500,

PRO412,GLY413,

VAL524,HIS525

ALA414,PRO415,

TRP528 ILE476,PHE478,HIS479

*Plant LDS was taken as 100% in each case.

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phase of these complexes. Results for RMSD of plantand fungal laccases complexed with monomer(Figure 3(A)) revealed that the difference in their RMSDwas not much significant suggesting that monomers areequally stable in both plant and fungal laccases. On theother hand, in the case of tetramer (Figure 3(B)),considerable difference between the RMSD of plant andfungal laccases was observed during the equilibriumphase (15–25 ns).

It is noteworthy that in case of monomer, the dockedcomplex of plant laccase stabilizes earlier than fungallaccase while, in case of tetramer, the docked complexof fungal laccase stabilizes much before plant laccase.Furthermore, a comparison of average RMSD of boththe fungal complexes revealed a decrease in averageRMSD from .3 to .25 for monomeric to tetrameric com-plexes, while for both the plant complexes, an increasein average RMSD from .35 (monomer) to .45 (tetramer)was observed. Therefore, based on these observations, itmay be suggested that the stability of plant laccase com-plex decreases with increasing the size of the ligninmodel compounds, while in case of fungal laccase, thestability of the complex behave in an opposite manneri.e. increase in stability with the size of the model com-pounds. Our result of MD simulation was found to be inconformity with the results obtained from dockinganalyses (Table 3).

We have also analyzed the interaction energies aswell as hydrogen bond interactions of both the plant andfungal laccases complexed with monomeric/teramericcompounds. The results are shown in Table 4. A com-parison of average interaction energies of monomerrevealed lower values for fungal laccase than that ofplant laccase, suggesting higher stability of fungallaccase with monomer. Similarly, the higher stability ofthe fungal laccase–monomer complex as compared tothat of plant was also evident from the analyses ofaverage number of hydrogen bonds per timeframe ofsimulation (Table 4). A similar conclusion can also bedrawn from the analyses of interaction energies andhydrogen bond interactions of the complexes of plantand fungal laccases with tetrameric compounds (Table 4).Thus, these results were in agreement with the results ofthe docking and MD simulation studies (docking scoresand RMSD trajectory of plant and fungal laccases withmonomer/tetramer).

In order to have an insight of the interactions occur-ring between the lignin model compounds at the activesite of laccases, in the three-dimensional space duringsimulation analyses, the snapshots of MD simulations ofmonomeric and tetrameric lignin model compounds withplant and fungal laccases were taken at different timeintervals and the results are shown in Figures 4–7. Thus,the snapshots at 0, 5, and 10 ns (Figures 4 and 5)

Figure 3. RMSDs of plant (red) and fungal (black) laccases complexed with monomer, sinapyl alcohol (A) and tetramer, guaiacylβ-O-4 syringyl β-β syringyl β-O-4 guaiacyl (B) during 25 ns MD simulation.

Table 4. Average interaction energies (kJ/mol) and average number of hydrogen bonds per time frame for various lignin modelcompounds during MD simulation (25 ns), with plant and fungal laccases at equilibrium phase.

Interaction energy

Plant laccase Fungal laccase

Monomer Tetramer Monomer Tetramer

Coulomb-SR (short range) −2.085 −7.15636 −3.662 −8.874Lennard-Jones-SR (short range) −127.398 −327.879 −141.981 −336.004Lennard-Jones-LR (long range) −8.350 −33.968 −9.901 −34.9373Average number of H-bonds per simulation timeframe .320 .733 .556 .978

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elucidate the movement of monomer towards the bindingsite and changes in the conformation of protein bindingcavity to accommodate the ligand. Further, the snapshotsat 15, 20, and 25 ns represent continuous stability of themonomer at the catalytic site for both plant and fungallaccases. The snapshots of plant laccase with tetramer(Figure 6) revealed that the ligand remains attached tothe laccase only through a small region at the bindingsite, and the major part of the tetramer remain unbound

and positioned outwards from the active site of theenzyme throughout the simulation period. On the otherhand, in the case of fungal laccase, the tetrameric ligandremained bound all along its length tightly to the enzymeactive site throughout the simulation period (Figure 7).

Thus, with regards to the dual function of lignin bio-synthesis and degradation exhibited by plant and fungallaccases, respectively, from MD simulation studies(Figures 4–7), it can be assumed that for lignin

Figure 4. Snapshots of plant laccase complexed with monomer sinapyl alcohol at different stages of MD simulation.

Figure 5. Snapshots of fungal laccase complexed with monomer sinapyl alcohol at different stages of MD simulation.

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degradation, the polymer must be able to be accommo-dated within the catalytic site of the enzyme which isobserved during the MD simulation of fungal laccasecomplexed with tetramer. The tetramer remains boundwithin the binding site of the enzyme and thus, allows theenzyme to catalyze the degradation reaction, unlike plant

laccase where the tetramer is unable to bind effectively atthe active site of the enzyme. Furthermore, the orientationof the bound tetramer with plant laccase (i.e. facing awayfrom the enzyme) is also suggestive of its participation inbiosynthesis of lignin in a manner comparable to that ofprotein biosynthesis (see supplementary S Video 1–4).

Figure 6. Snapshots of plant laccase complexed with tetramer guaiacyl β-O-4 syringyl β-β syringyl β-O-4 guaiacyl at different stagesof MD simulation.

Figure 7. Snapshots of fungal laccase complexed with tetramer guaiacyl β-O-4 syringyl β-β syringyl β-O-4 guaiacyl at differentstages of MD simulation.

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3.5. Binding free-energy analyses

In order to have deeper insight into the stability of theligands to the target with regards to the differential

enzymatic catalysis by plant and fungal laccases per-taining to lignin biosynthesis and degradation, the bind-ing free energies of complexes of the lignin model

Table 5. Binding energies (kJ/mol) for various lignin model compounds complexed with plant and fungal laccases computed usingMM/PBSA during 15–25 ns (equilibrium phase) of MD simulation trajectory.

Energy components

Plant laccase Fungal laccase

Monomer Tetramer Monomer Tetramer

ΔEelea −4.296 −6.188 −6.018 −3.638

ΔEvdwb −133.959 −369.831 −153.363 −399.941

ΔGpolc 30.639 82.334 31.645 59.244

ΔGnonpold −12.353 −30.428 −13.091 −28.669

ΔGbinde −119.969 −324.113 −140.827 −373.004

aElectrostatic interaction energies.bvan der Waals interaction energies.cPolar contributions to the solvation free energy.dNonpolar contributions to the solvation free energy.eBinding energies.

Table 6. Summary of the results of present in silico analyses showing comparison of salient features of the plant and fungallaccases.

In-silico approaches Plant laccase (Populus trichocarpa) Fungal laccase (Trametes versicolor)

Cooper binding motifanalysis (H-X-H motifs)

Motif 1-X = TRP Motif 1-X = TRPMotif 2-X = ALA Motif 2-X = SERMotif 3-X = LEU Motif 3-X = LEUMotif 4-X = CYS Motif 4-X = CYS

Pairwise sequencealignment

27.6% sequence identity and 45.9% sequence similarity

Homology modeling C-terminal region forms an extended structuregenerating a movable plug which helps in trapping ofthe oxygen

C-terminal region forms a coiled structureimparting rigidity in movement and allowingfree flow of oxygen.

Superimposition RMSD of 7.25 Å (not so significant similarity)Molecular docking Larger compounds such as trimers and tetramers

interact less efficientlyLarger compounds such as trimers andtetramers interact more efficiently.

Interacting residues(common for all ligninmodel compounds)

MET287, ASP288, ASN333, PRO454, GLU455,SER456, THR500, HIS525

LEU185, ASP227, ASN229, PHE260,SER285, PHE286, GLY413, ALA414,PRO415, ILE476, HIS479

Molecular dynamicssimulation

� Docked complex with monomer stabilizesearlier (10 ns)

� Increase in average RMSD from .35 (monomer)to .45 nm (tetramer)

� Stability of docked complex decreases withincrease in the size of the lignin modelcompounds

� Tetrameric ligand remains attached only througha small region at the binding site and major partremains unbound and positioned outwards fromthe active site of the enzyme

� Docked complex with tetramer stabilizesearlier (5 ns)

� Decrease in average RMSD from .3(monomer) to .25 nm (tetramer)

� Stability of docked complex increaseswith the size of the lignin modelcompounds

� Tetrameric ligand remained bound allalong its length tightly to the enzymeactive site

MM/PBSA binding freeenergy (kJ/mol)

� Monomer: −119.969� Tetramer: −324.113

� Monomer: −140.827� Tetramer: −373.004

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compounds with those of plant and fungal laccaseswere computed using MM/PBSA method. Results ofthe analyses for the 15–25 ns (equilibrium phase) ofMD simulation are presented in Table 5. From the datapresented in Table 5, it is evident that fungal laccase,complexed with tetrameric lignin ligand exhibited abinding energy (ΔGbind) of −232.177 kJ/mol lesser thanthat of fungal laccase, complexed with monomeric lig-nin ligand. In comparison to this, in case of plant lac-case, a net decrease of binding energy of −204.144 kJ/mol between monomeric and tetrameric complexes wasobserved. Furthermore, the difference between bindingenergies of plant and fungal laccases, complexed withmonomeric lignin ligand, was −20.858 kJ/mol whilethose of plant and fungal laccases, complexed with tet-rameric lignin ligand, was −48.891 kJ/mol. Thus, morethan twofolds lesser binding energy for tetrameric lig-nin ligand complexed with plant/fungal laccases, ascompared to those of monomeric ligands, wasobserved. Based on the individual contributions of thevarious components of energy towards the bindingenergy (ΔGbind), it may also be suggested that thehigher stability of the tetrameric complexes may be dueto the van der Waals and polar solvation energies.Thus, based on these observations, it may be suggestedthat the stability of fungal laccase complexes were sig-nificantly higher than those of plant laccase complexes,and this stability increases in order of increasing thelength of the lignin model compounds. Overall,MM/PBSA binding free-energy analyses corroboratedwell with the results of molecular docking and dynam-ics simulation analyses, and revealed significantly lowerbinding energy for lignin model compounds, in propor-tion to their size, for fungal laccase than those of plantlaccase. Results of present in silico analyses showingcomparison of salient features of the plant and fungallaccases are summarized in Table 6.

4. Conclusions

Laccases due to their ability to catalyze the oxidation ofa range of compounds makes them more versatile withregards to their function as well as industrial applica-tions. Among the diverse functions of laccases, the ligni-fication/delignification has been considered as one of thevery significant function because of their involvement invarious industrial applications such as pulp and papermanufacturing, biobleaching, bioenergy production andbiofuel. In spite of the similar basic mechanism for lac-case-mediated catalysis, the basis of the dual function ofplant and fungal laccases in lignin biosynthesis anddegradation, respectively, has not yet been suggestedunequivocally. The present study is an attempt toinvestigate the unambiguous basis of the dual functions

mediated by plant and fungal laccases with regards tolignin biosynthesis and degradation, respectively, usingin silico approaches at sequence, structure, as well asmolecular dynamic interaction levels. Thus, the results ofmodeling, docking, and simulation analyses of plant andfungal laccases, taken together, namely structural differ-ences at the C-terminal region, differences in the inter-acting residues of the binding site, different bindingmodes of tetramer at the active site, differences in theRMSD, interaction energies, hydrogen bond interactions,and binding free-energy analyses during MD simulation,provide sufficient evidences for the better understandingof the mechanism of lignification and delignification byplant and fungal laccases, respectively. Thus, the presentstudy provided useful tool to understand the molecularbasis of lignin synthesis and degradation, and would,in future, help to identify strategies to modify laccasestructure in plants and fungi to improve ligninbiosynthesis and biodegradability, respectively.

Supplementary Material

The supplementary material for this paper is availableonline at http://dx.doi.10.1080/07391102.2014.975282.

FundingFinancial supports from the Department of Biotechnology,Govt. of India, New Delhi, under the BIF program, the Depart-ment of Higher Education, Govt. of U.P., under the Center ofExcellence in Bioinformatics program, University Grants Com-mission, New Delhi, under UGC-DSK Fellowship Scheme (toNJ), and from the Department of Science & Technology, Govt.of India, New Delhi, under DST-INSPIRE Fellowship (to SS)& DST-PURSE programs are gratefully acknowledged.

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