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

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

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Manika Awasthi , Nivedita Jaiswal , Swati Singh , Veda P. Pandey & U.N. Dwivedi

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Bioinformatics Infrastructure Facility, Center of Excellence in Bioinformatics, Department of Biochemistry, University of Lucknow, Lucknow -226007, India Accepted author version posted online: 10 Oct 2014.

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

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Publisher: Taylor & Francis Journal: Journal of Biomolecular Structure and Dynamics

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DOI: http://dx.doi.org/10.1080/07391102.2014.975282

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

Manika Awasthi, Nivedita Jaiswal, Swati Singh, Veda P. Pandey and U.N. Dwivedi* Bioinformatics Infrastructure Facility, Center of Excellence in Bioinformatics Department of Biochemistry, University of Lucknow, Lucknow -226007, India

Running title: Molecular docking and dynamics simulation of plant and fungal laccases.

*Corresponding author Email: [email protected] 1


Telefax: 91-522-2740132

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Abstract Laccase, widely distributed in bacteria, fungi and plants, catalyzes the oxidation of wide range of compounds. With regards to one of the important physiological functions, plant laccases are considered to catalyze lignin biosynthesis while fungal laccases are considered for lignin degradation. The present study was undertaken to explain this dual function of laccases using in


silico molecular docking and dynamics simulation approaches. Modeling and superimposition analyses 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 significantly higher binding efficiency for lignin model compounds, in proportion to their size, for fungal laccase as compared to that of plant laccase. Residues interacting with the model compounds at the respective enzyme active sites were found to be in conformity with their role in lignin biosynthesis and degradation. Molecular dynamics simulation analyses for the stability of docked complexes of plant and fungal laccases with lignin model compounds revealed that tetrameric lignin model compound remains attached to the active site of fungal laccase, throughout the simulation period while, it protrudes outwards from the active site of plant laccase. Stability of these complexes was further analyzed on the basis of binding energy which revealed significantly higher stability of fungal laccase with tetrameric compound than those of plant. The overall data suggested a situation favorable for degradation of lignin polymer by fungal laccase while its synthesis by plant laccase.

Keywords: Homology modeling, laccase, lignin biosynthesis and degradation, molecular docking, molecular dynamics simulation. 3

1. Introduction Laccases (benzenediol: oxygen oxidoreductase; EC 1.10.3.2) are multicopper oxidases, widely distributed in bacteria, fungi, higher plants and insects (Hoegger, Kilaru, James, Thacker, & Kues, 2006). They belong to blue copper oxidase superfamily and have ability to catalyze oxidation of a wide range of compounds such as substituted phenols, diamines, aromatic amines,


thiols and even some inorganic compounds. The ability of laccases, to catalyze the oxidation of a range of compounds, makes them more versatile with regards to their function as well as industrial

applications.

Thus,

the

diverse

functions

mediated

by

laccases

include

polymerization/depolymerization of lignin, fungal pathogenesis, wound healing, sclerotization, morphogenesis, sporulation, pigmentation, fruiting body formation, melanin formation, endospore coat protein synthesis etc. (Dwivedi, Singh, Pandey, & Kumar, 2011). Among these diverse functions of laccases, the lignification/delignification has been considered as one of the very significant function because of their involvement in various industrial applications such as pulp and paper manufacturing, biobleaching, bioenergy production, biomass conversion, biofuel, removal of a large number of environmental pollutants, such as alkenes, chlorophenols, dyes, herbicides, polycyclic aromatic hydrocarbons and benzopyrene etc. (Couto, Herrera, & Toca, 2006; Gianfreda, Xu, & Bollag, 1999; Jaiswal, Pandey, & Dwivedi, 2014a; Lange, & Grell, 2014). In plants, the lignification process encompasses the polymerization of monolignols via their dehydrogenation followed by combinatorial free radical coupling by a couple of enzymes including laccases in the cell wall. Based on experimental studies, it has been reported that laccases from several plant species efficiently oxidize monolignols to dehydrogenative polymers (Bao, O'malley, Whetten, & Sederoff, 1993; Chabanet et al., 1994; Davin, Bedgar, Katayama, & 4

Lewis, 1992; Driouich, Lainel, Vian, & Faye, 1992; Liu, Dean, Friedman, & Eriksson, 1994; McDougall, & Morrison lan, 1996; Sterjiades, Dean, & Eriksson, 1992). Expression of laccase, predominantly in the secondary xylem, have been reported from trees like Populus trichocarpa (Ranocha et al., 1999) and Pinus taeda (Sato, Wuli, Sederoff, & Whetten, 2001), suggesting its role in lignin biosynthesis in plants. Evidence for participation of laccase in lignin biosynthesis


has also been reported on the basis of plants transformation studies 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 mediated breakdown of lignin polymer and oxidative degradation of the side chains of the p-hydroxyphenyl (H), guaiacyl (G) and syringyl (S) lignin units, releasing a set of phenolic compounds (acids, ketones and aldehydes) (Camarero, Galletti, & Martinez, 1994). The various products of these oxidative degradation reactions have also been reported to act like mediators (endogenous) and thereby further promote the oxidative degradation of the lignin polymer (Camarero, Ibarra, Martınez, & Martı´nez, 2005; Cañas et al., 2007; Eggert, Temp, Dean, & Eriksson, 1996; Nousiainen, Maijala, Hatakka, Martínez, & Holzforschung, 2009; Torres-Duarte, Roman, Tinoco, & Vazquez-Duhalt, 2009). The degradation of the lignin model compounds namely, syringyl glycol-β-guaiacyl ether and syringyl type β-aryl ether by laccase from Polyporus versicolor and Stereum frustulatum have also been demonstrated (Kirk, Harkin, & Cowling, 1968; Wariishi, Morohoshi, & Haraguchi, 1987). Moreover, a diminished lignin degrading ability in laccase-minus mutants of Sporotrichzum puluerulentum while enhanced lignolytic activity in laccase-plus mutants have been reported (Ander & Eriksson, 1976). The fundamental molecular architecture of laccase copper centre, distributed into three redox sites namely, T1, T2 and T3 sites, at the active site of laccases, is quite similar in both 5

plant and fungal laccases (Claus, 2004). Despite of this similarity in molecular architecture, plant and fungal laccases have been reported to possess wide phylogenetic, physicochemical as well as functional diversity (Colaneri, & Vitali, 2014; Dwivedi et al., 2011; Satpathy, Behera, Padhi, & Guru, 2013). Thus, in the light of the reports stated above, it is evident that plant laccases are involved


in lignin biosynthesis while fungal laccases in lignin degradation or depolymerization. However, the basis of the dual function of laccases catalyzing lignin biosynthesis (plants) and degradation (fungi), has not yet been elucidated unequivocally. Thus, few reports have shown that redox potential of laccase play a crucial role in deciding the fate of the reaction i.e. whether lignification or delignification. Fungal laccases due to their higher redox potential (upto +800 mV) are capable of acting on both the phenolic and non-phenolic subunits of lignin thereby, playing a role in lignin degradation. Plant laccases polymerize lignin by coupling of the phenoxy radicals due to its 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 different amino acid residues at the T1 copper site. Plant laccases have a methionine residue in addition to one cysteine and two histidine residues to construct the T1 copper site (Nitta, Kataoka, & Sakurai, 2002), while in fungal laccases phenylalanine is found to play the role (Morozova, Shumakovich, Gorbacheva, Shleev, & Yaropolov, 2007). Xu et al. (1998) observed that Trametes versicolor laccase, having high redox potential (800 mV), have a phenylalanine residue instead of methionine and predicted that it might be responsible for the high redox potential. Methionine forms a strong axial bond to T1 copper while phenyalanine do not bind to copper and thus increase hydrophobicity surrounding T1 copper. Due to absence of bonds to the axial ligand, the Cu-Scys bond is much stronger which accounts for the increase in redox potential of fungal 6

laccases. pH dependences of fungal and plant laccases has also been suggested as one of the basis for this dual action of laccases in lignin degradation or synthesis (Gorbacheva et al., 2008; Madhavi & Lele, 2009). Thus, fungal laccases exhibit low pH optima which may be due to their adaptation to grow under acidic conditions, while plant laccases being intracellular have their pH optima nearer to the physiological range. Thus, the differences in pH optima might be suggested


to be linked to this dual function of laccases. In addition, the overall three dimensional structure of laccase, leading to altered microenvironment at the active site of the enzyme, has also been suggested as the basis of the dual action of lignin biosynthesis and degradation catalyzed by the two laccases. Thus, based on the crystallographic studies of two distinct types of fungal laccases, one involved in lignin degradation (Trametes versicolor [basidiomycetes]) while other involved in lignin biosynthesis (Melanocarpus albomyces [ascomycetes]) (similar to that of plant laccase), Hakulinen et al. (2002) have reported that in the laccase of Trametes versicolor, oxygen enters in the tri-nuclear cluster through an open tunnel whereas, in laccase of Melanocarpus albomyces, the C-terminus forms a movable plug which can block this access and trap the oxygen. This C-terminal blockage of the M. albomyces laccases significantly reduces the speed of the free inflow of O2 and release of water molecules thereby, make the surrounding environment more appropriate for polymerization while, in case of T. versicolor laccases, the rapid exchange of O2 and water does not allow for the build-up of free radicals in the microenvironment and thus, avoiding polymerization. Thus, the authors have proposed that the architectural differences at the Cterminal end of M. albomyces and T. versicolor laccases might be responsible for their role in lignification and delignification, respectively.

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A comparison of some of the salient features of plant and fungal laccases, illustrating broader differences among them, are presented in Table 1. The present work was initiated with the objective of providing a newer basis of the yet unequivocally explained dual role of lignin biosynthesis and degradation exhibited by plant and fungal laccases. With this objective, using relevant bioinformatical tools, which is of both


academic as well as of practical interest looking to the wide application of laccases. Thus, laccases from two representative classes belonging to each of plant and fungi have been analyzed at the levels of protein sequence, 3D structure as well as molecular dynamics interactions using relevant in-silico tools. For copper binding motif analyses, thirty protein sequences each of the two classes of laccase (plant and fungal) were retrieved and analyzed. 3D-structures of Populus trichocarpa and Trametes versicolor laccase, representatives of each plant and fungal laccase, respectively, were modeled and docked with various lignin model compounds of different complexity levels, to compare the active site interactions in both the representative classes of laccases. The docked complexes were further subjected to 25 nanoseconds (ns) molecular dynamics simulations to investigate and compare the binding efficiency and stability of various compounds with the modeled laccases. Results of the in silico analyses were validated and discussed in light of data available from experimental studies. 2. Materials and methods 2.1. Sequence analysis Thirty homologous laccase protein sequences, each from plant and fungi, were selected from NCBI database (http://www.ncbi.nlm.nih.gov). The multiple sequence alignment for each of plant and fungal group of laccase sequences were performed by COBALT MSA tool on

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default parameters (Papadopoulos & Agarwala, 2007). Copper binding motif analyses of the laccase sequences 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 Populus trichocarpa while that of from a fungi Tramates versicolor, as respective representative classes, were selected. In order to


select a template for homology modeling of laccases, BLASTP (Altschul, Gish, Miller, Myers, & Lipman, 1990) analyses were performed. Based on high similarity score, crystal structure of ascorbate oxidase (1AOZ) and laccase complexed with 2, 5-xylidine (1KYA) were used as template structures for modeling of plant and fungal laccases, respectively. Protein sequences of both the laccases and their corresponding templates were aligned accurately and 3D models were generated using ‘Build Homology Models’ module of Discovery Studio 3.5 (DS) (Accelrys Software Inc.). The ‘Loop Refinement’ and ‘Side-Chain Refinement’ modules were used for refinement of initial 3D models of laccase. Finally, the energy minimizations were performed using CHARMM27 forcefield by 500-step steepest-descent (SD) minimization followed by conjugate gradient (CG) minimization. The final laccase models were further validated using ‘Modeler’ module of DS for protein verification and tested for stereo-chemical accuracy with the ‘Procheck’ online program on SAVES server (http://nihserver.mbi.ucla.edu/SAVES/). Residue compatibility was assessed by the ‘Profile-3D’ module in DS. Modeled plant and fungal laccases were further superimposed on each 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 guaiacyl β-O-4 syringyl β-β syringyl β-O-4 guaiacyl (tetramer) were selected from NMR database of lignin and cell wall 9

model compounds (S. Ralph, J. Ralph, & Landucci, 2004) and their structures were sketched (Table 3). Energy minimization was performed using CHARMm forcefield and conformations were generated for each compound using BEST algorithm. 2.4. Molecular docking of laccases with various lignin model compounds The active sites of the selected representative classes of plant and fungal laccases were


predicted based on the PDB site records using ‘Define and Edit Binding Site module’ and subsequently docking of the selected lignin model compounds was performed using ‘LibDock module’ of DS. Docking interactions between laccases and selected lignin model compounds were analyzed with the help of the ‘Analyze complexes’ module of DS. Based on the docking score, the best conformation of each compound was selected for further analyses. 2.5. Molecular dynamics simulations of docked complexes Stability of the docked complexes and interactions of plant and fungal laccases with the selected lignin model compounds were investigated through 25 ns of molecular dynamics (MD) simulations using GROMACS 4.5.5 package (Pronk et al., 2013) with GROMOS96 43a1 force field. The docking poses of laccase with respective lignin model compounds were prepared for MD simulation through mild minimization and solvation within a water filled 3D cube of 1 Å spacing using simple point charge (SPC), a 3-point model for water. A leap-frog time integration algorithm was used for integrating Newton's equations of motion. System was neutralized and further minimized. The complex structure was heated to 300K and equilibrated for 100 picoseconds (ps) in NVT ensemble and another 100 ps in NPT ensemble. After heating and equilibration, the laccase-lignin model compound complex was subjected to production run of 25 ns in NPT ensemble. PRODRG web server (SchuÈttelkopf & van Aalten, 2004) was used to generate topologies and coordinates of ligands. Default values of GROMACS were assigned for 10

determination of hydrogen bonding interactions and interaction energies between laccase and lignin model compounds. 2.6. Binding free energy analyses The binding free energies of the complexes between laccases and various lignin model compounds, during the MD simulation analyses, were computed using g_mmpbsa tool of


GROMACS (Kumari, Kumar, OSDD consortium, & Lynn, 2014) based on the molecular mechanics/Poisson-Boltzman surface area (MM/PBSA) method (Kollman et al., 2000). The binding energy calculations were performed for 1000 snapshots taken at an interval 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 Multiple sequence alignment (MSA) for thirty each of plant and fungal laccases were done. An in depth analysis of MSA results revealed the presence of several conserved stretches of 20-30 amino-acid residues. The position of these stretches in alignment lies in range of 158178 and 649-669 for fungi and 170-190 and 658-678 for plants. These two stretches of 21 aminoacid residues each, shows highest degree 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 conserved stretches, a search was made against PROSITE database, which revealed the match of these conserved stretches with multicopper oxidase pattern (PS00079 and PS00080) of PROSITE. The pattern consisted of four H-X-H motifs (copper binding region) which form copper interaction site with highly conserved histidine residues (Supplementary Figures S1 and S2). Valderrama, Oliver, Medrano-Soto, and Vazquez-Duhalt (2003) have reported that the highly conserved histidine residues interact with copper ion during laccase mediated catalysis. 11

The prominent conservation at multi-copper domains among laccases has been suggested to play key role in laccase mediated catalysis as well as in its evolution (Messerschmidt & Huber, 1990; Solomon, Sundaram, & Machonkin, 1996). Comparative analysis of H-X-H motif shows a remarkable difference at position X within group as well as outside the group. In the first H-X-H motif, tryptophan was found to be highly


conserved in both plants and basidiomycetes, however, in ascomycetes a phenylalanine was found. The second H-X-H motif shows higher conservation within group, but more variation out of group. In basidiomycetes, serine shows higher conservation while ascomycetes and plants are characterized by alanine. The third and fourth H-X-H motif showed highest conservation within the group as well as outside the group as compared to first two motifs explained above. The X residue of third motif (position between T2 and T3) represents leucine in plants and basidiomycetes, while lysine in ascomycetes. The fourth motif, characterized by HCH, showed 100% conservation within as well as outside the groups. These observed differences may be significant from the point of view of enzyme catalysis with regards to lignin biosynthesis/degradation. 3.2. 3D structure modeling of plant and fungal laccases The homology modeling generated five models for each of selected plant and fungal laccases with different DOPE scores. The respective models with minimum DOPE scores (Figure 1) were selected for further analyses. The modeled laccases with minimum DOPE scores were further validated using Modeler, Procheck and Profile-3D and were found to be reliable for interpretation of structure-function relationships (Table 2). The analyses of 3D structures of the two laccases revealed a similar folding having three sequentially arranged cupredoxin-like domains. These domains are formed by β-sheets and β-strands, arranged in sandwich 12

conformation (Lindley, 2001). The results were in agreement with those reported for similar representative laccases from X-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 laccases in the present study revealed prominent differences in the folding of C-terminal region. The role of C-terminal residues of


fungal laccases in enzyme catalysis and stability has been investigated in detail by Bleve et al. (2013) by performing various deletions as well as substitutions using site-directed mutagenesis. The C-terminal region of plant laccase formed an extended structure which could easily form a movable plug as has been reported for M. albomyces (Hakulinen et al., 2002) where it has been suggested to help in trapping of the oxygen during laccase mediated lignin biosynthesis. On the other hand, in the case of fungal laccase the C-terminal part of the protein formed a coiled structure, imparting rigidity in the movement and thereby allowing free flow of oxygen. In agreement to our findings, Hakulinen et al. (2002) have also reported a distinct difference at the C-terminal end in the crystal structure of M. albomyces and T. versicolor laccase and suggested that this difference may be the reason for the dual behavior of laccase. Thus, in present study plant laccase was found to have similar 3D structure as proposed for M. albomyces (Hakulinen et al., 2002). Since, plant laccase possessed similar function to M. albomyces laccase i.e. polymerization of monolignols therefore, the mechanism of free radical generation through Cterminal blockage of O2 could also be applied to plant laccase. Moreover, M. albomyces laccase also exhibited low redox potential similar to plant laccase. 3.2.1. Superimposition analysis of modeled laccases Superimposition analysis of modeled plant and fungal laccases revealed a root mean square deviation (RMSD) of 7.25 Å with 454 overlapping residues (Figure 2) suggesting a not so 13

significant similarity at 3D structure level among these laccases. Furthermore, pair-wise sequence alignment of primary sequences of the two modeled laccases revealed 27.6% sequence identity and 45.9% sequence similarity with highly conserved copper binding motifs. 3.3. Docking studies of modeled laccases with lignin substrates Molecular docking of both plant and fungal laccases with various lignin model


compounds was done using DS. Results are shown in Table 3. Results of docking analyses revealed that binding efficiency (based on docking scores) of lignin model compounds increased with the size of the model compounds in both fungal as well as plant laccases, however, the degree of the increase in binding efficiency was found to be significantly higher for fungal laccase as compared to that of plant laccase. Thus, the ratio of LibDock scores (LDS) of fungal laccase to that of plant laccase were found to be 1.419 for monomer, 1.457 for dimer, 1.556 for trimer and 2.037 for tetramer corresponding to a fold increase of 41.91% for monomer, 45.73% for dimer, 55.64% for trimer and 103.71% for tetramer (Table 3). Thus, based on the present study, it could be concluded that larger compounds such as trimers and tetramers interact much more efficiently with the fungal laccase than that of plant laccase suggesting a possible reason for dual activity exhibited by these laccases. 3.3.1. Regiospecificity of binding of various lignin model compounds to plant and fungal laccases Regiospecificity of binding of various lignin model compounds at the active site of plant and fungal laccases was compared. Results depicting various interacting residues are presented in Table 3. Plant laccase was found to interact with a total of 11, 16, 23 and 21 amino acid residues in the case of monomer, dimer, trimer and tetramer, respectively, out of which, 8 residues namely, MET287, ASP288, ASN333, PRO454, GLU455, SER456, THR500 and 14

HIS525 were found to be common in the binding of all the model compounds. Fungal laccase was found to interact with a larger number of amino acid residues i.e. 14, 17, 23 and 23 in the case of 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 to be common in the binding of all the model compounds. In agreement


with our results, Kallio et al. (2009) while elucidating the crystal structure of M. albomyces laccase co-crystallized with a lignin model compound have shown involvement of seven residues, namely ALA, PRO, GLU, LEU, PHE, TRP and HIS in the binding of lignin model compound at the active site of laccase. The same set of residues was found to interact with the lignin model compounds in the present study. Histidine residues have been shown to interact strongly with aromatic rings in a size dependent manner, through π–π stacking interactions with aromatic rings (such as lignin model compounds, used in present study), leading to catalysis (Rocha, Ramalho, Caetano, & da Cunha, 2013). With regards to dual function of lignin biosynthesis and degradation exhibited by plant and fungal laccases, respectively, in the present study, the fungal laccases which have high redox potential was found to involve phenylalanine in the binding of all the lignin model compounds, while in case of plant laccases which are characterized by a low redox potential, a methionine was found to be involved in binding of these model compounds. In agreement to the present analyses, Morozova et al. (2007) have reported that the high-potential laccases have a phenylalanine residue as an axial ligand of the T1 site copper while in low-potential laccases this role is played by methionine residue. Based on site-directed mutagenesis, Xu (1996) has also reported that laccases, harboring PHE at Type I copper site, exhibited high redox potential, whereas laccases with MET exhibited low redox potential. It has also been suggested that ASP 15

residue at the binding site of fungal laccase plays a crucial role in proton abstraction from the substrate, but in plant laccase ASN residue instead of ASP, perform the same role (Frasconi, Favero, Harry, Anu, & Franco, 2010; Madzak et al., 2006). Similar to these reports, in our study also an ASP227 has been observed at the active site of fungal laccase, while that of ASN228 in plant laccase. Furthermore, the binding of monomer, trimer and tetramer to plant and fungal


laccases exhibited conformational changes also (Supplementary Figure S3), thereby suggesting the basis for differences in LibDock scores and this might be the possible reason for the dual catalytic differences in plant and fungal laccases. 3.4. Molecular dynamics simulation analysis For MD simulation studies two types of lignin model compounds, at the two extremes, namely a monomer (sinapyl alcohol) and a tetramer (guaiacyl β-O-4 syringyl β- β syringyl β-O-4 guaiacyl) were selected. Thus, a total of four 25 ns MD simulations, two each for monomer and tetramer with plant and fungal laccases, were done. Results for root mean square deviation (RMSD) for plant and fungal laccases complexed with monomeric and tetrameric lignin model compounds are shown in Figure 3. It is noteworthy, that both plant and fungal laccases complexed with monomeric/tetrameric lignin model compounds maintained equilibrium between 15-25 ns of simulation. All further analyses were performed during this equilibrium phase of these complexes. Results for RMSD of plant and fungal laccases complexed with monomer (Figure 3A) revealed that the difference in their RMSD was not much significant suggesting that monomers are equally stable in both plant and fungal laccases. On the other hand, in the case of tetramer (Figure 3B), considerable difference between the RMSD of plant and fungal laccases was observed during the equilibrium phase (15-25 ns).

16

It is noteworthy that, in case of monomer the docked complex of plant laccase stabilizes earlier than fungal laccase while in case of tetramer the docked complex of fungal laccase stabilizes much before plant laccase. Furthermore, a comparison of average RMSD of both the fungal complexes revealed a decrease in average RMSD from 0.3 to 0.25 for monomeric to tetrameric complexes, while for both the plant complexes an increase in average RMSD from


0.35 (monomer) to 0.45 (tetramer) were observed. Therefore, based on these observations, it may be suggested that the stability of plant laccase complex decreases with increasing the size of the lignin model compounds while in case of fungal laccase the stability of the complex behave in an opposite manner i.e. increase in stability with the size of the model compounds. Our result of MD simulation was found to be in conformity with the results obtained from docking analyses (Table 3). We have also analyzed the interaction energies as well as hydrogen-bond interactions of both the plant and fungal laccases complexed with monomeric/terameric compounds. The results are shown in Table 4. A comparison of average interaction energies of monomer revealed lower values for fungal laccase than that of plant laccase, suggesting higher stability of fungal laccase with monomer. Similarly, the higher stability of the fungal laccase-monomer complex as compared to that of plant was also evident from the analyses of average number of hydrogenbonds per timeframe of simulation (Table 4). A similar conclusion can also be drawn from the analyses of interaction energies and hydrogen-bond interactions of the complexes of plant and fungal laccases with tetrameric compounds (Table 4). Thus, these results were in agreement with the results of the docking and MD simulation studies (docking scores and RMSD trajectory of plant and fungal laccases with monomer/tetramer).

17

In order to have an insight of the interactions occurring between the lignin model compounds at the active site of laccases, in the three-dimensional space, during simulation analyses, the snapshots of MD simulations of monomeric and tetrameric lignin model compounds with plant and fungal laccases, were taken at different time intervals and the results are shown in Figures 4-7. Thus, the snapshots at 0, 5 and 10 ns (Figures 4 and 5) elucidate the


movement of monomer towards the binding site and changes in the conformation of protein binding cavity to accommodate the ligand. Further, the snapshots at 15, 20 and 25 ns represent continuous stability of the monomer at the catalytic site for both plant and fungal laccases. The snapshots of plant laccase with tetramer (Figure 6) revealed that the ligand remains attached to the laccase only through a small region at the binding site and the major part of the tetramer remain unbound and positioned outwards from the active site of the enzyme throughout the simulation period. On the other hand, in the case of fungal laccase, the tetrameric ligand remained bound all along its length tightly to the enzyme active site throughout the simulation period (Figure 7). Thus, with regards to the dual function of lignin biosynthesis and degradation exhibited by plant and fungal laccases, respectively, from MD simulation studies (Figures 4-7), it can be assumed that for lignin degradation, the polymer must be able to be accommodated within the catalytic site of the enzyme which is observed during the MD simulation of fungal laccase complexed with tetramer. The tetramer remains bound within the binding site of the enzyme and thus, allows the enzyme to catalyze the degradation reaction, unlike plant laccase where the tetramer is unable to bind effectively at the active site of the enzyme. Furthermore, the orientation of the bound tetramer with plant laccase (i.e. facing away from the enzyme) is also

18

suggestive of its participation in biosynthesis of lignin in a manner comparable to that of protein biosynthesis (See supplementary S Video 1-4). 3.5. Binding free energy analyses In order to have deeper insight into the stability of the ligands to the target with regards to the differential enzymatic catalysis by plant and fungal laccases pertaining to lignin biosynthesis


and degradation, the binding free energies of complexes of the lignin model compounds with those of plant and fungal laccases were computed using MM/PBSA method. Results of the analyses for the 15-25 ns (equilibrium phase) of MD simulation are presented in Table 5. From the data presented in Table 5, it is evident that binding energy of fungal laccase, complexed with tetrameric lignin ligand exhibited a binding energy (ΔGbind) of -232.177 kJ/mol lesser than that of fungal laccase, complexed with monomeric lignin ligand. In comparison to this, in case of plant laccase a net decrease of binding energy of -204.144 kJ/mol between monomeric to tetrameric complexes was observed. Furthermore, the difference between binding energies of plant and fungal laccases, complexed with monomeric lignin ligand, was -20.858 kJ/mol while those of plant and fungal laccases, complexed with tetrameric lignin ligand, was -48.891 kJ/mol. Thus, more than two-folds lesser binding energy for tetrameric lignin ligand complexed with plant/fungal laccases, as compared to those of monomeric ligands, was observed. Based on the individual contributions of the various components of energy towards the binding energy (ΔGbind), it may also be suggested that the higher stability of the tetrameric complexes may be due to the van der Waals and polar solvation energies which was significantly higher for tetrameric complexes than those of monomeric ones. Thus, based on these observations, it may be suggested that the stability of fungal laccase complexes were significantly higher than those of plant laccase complexes and this stability increases in order of increasing the length of the 19

lignin model compounds. Overall, MM/PBSA binding free energy analyses corroborated well with the results of molecular docking and dynamics simulation analyses and revealed significantly lower binding energy for lignin model compounds, in proportion to their size, for fungal laccase than those of plant laccase. Results of present in-silico analyses showing comparison of salient features of the plant and fungal laccases are summarized in Table 6.


4. Conclusions Laccases due to their ability to catalyze the oxidation of a range of compounds makes them more versatile with regards to their function as well as industrial applications. Among the diverse functions of laccases, the lignification/delignification has been considered as one of the very significant function because of their involvement in various industrial applications such as pulp and paper manufacturing, biobleaching, bioenergy production, biofuel, etc. In spite of the similar basic mechanism for laccase mediated catalysis, the basis of the dual function of plant and fungal laccases in lignin biosynthesis and degradation, respectively, have not yet been suggested unequivocally. The present study is an attempt to investigate the unambiguous basis of the dual functions mediated by plant and fungal laccases with regards to lignin biosynthesis and degradation, respectively, using in-silico approaches at sequence, structure as well as molecular dynamic interaction levels. Thus, the results of modeling, docking and simulation analyses of plant and fungal laccases, taken together, namely, structural differences at the C-terminal region, differences in the interacting residues of the binding site, different binding modes of tetramer at the active site, differences in the RMSD, interaction energies, hydrogen-bond interactions and binding free energy analyses during MD simulation, provide sufficient evidences for the better understanding of the mechanism of lignification and delignification by plant and fungal laccases, respectively. Thus, the present study provided useful tool to understand the molecular basis of 20

lignin synthesis and degradation and would, in future, help to identify strategies to modify laccase structure in plants and fungi to improve lignin biosynthesis and biodegradability, respectively.

Acknowledgements


Financial supports from the Department of Biotechnology, Govt. of India, New Delhi, under the BIF programme, the Department of Higher Education, Govt. of U.P., under the Center of Excellence in Bioinformatics programme, University Grants Commission, New Delhi, under UGC-DSK Fellowship Scheme (to NJ) and from the Department of Science & Technology, Govt. of India, New Delhi, under DST-INSPIRE Fellowship (to SS) & DST-PURSE programmes are gratefully acknowledged.

21

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Figure captions Figure 1. Three dimensional structures of plant (A) and fungal (B) laccases generated by homology modeling. Figure 2. 3D superimposition of modeled structures of plant (green) and fungal (red) laccases. Figure 3. Root mean square deviations of plant (red) and fungal (black) laccases complexed with


monomer, sinapyl alcohol (A) and tetramer, guaiacyl β-O-4 syringyl β- β syringyl β-O-4 guaiacyl (B) during 25 ns MD simulation. 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. Figure 6. Snapshots of plant laccase complexed with tetramer guaiacyl β-O-4 syringyl β- β syringyl β-O-4 guaiacyl at different stages of MD simulation. Figure 7. Snapshots of fungal laccase complexed with tetramer guaiacyl β-O-4 syringyl β- β syringyl β-O-4 guaiacyl at different stages of MD simulation. Figure captions for supplementary figures: Figure S1. Extracted fragments from multiple sequence alignment of the plant dataset. Consensus residues are marked with an asterisk. Copper interacting histidine residues are highlighted in bold font. T1, T2 and T3 represent the redox sites containing copper atoms. Figure S2. Extracted fragments from the multiple sequence alignment of the fungal dataset. Consensus residues are marked with an asterisk. Copper interacting histidine residues are highlighted in bold font. T1, T2 and T3 represent the redox sites containing copper atoms.

30

Figure S3. 2D view showing conformational changes occurring during the binding of various lignin model compounds at the active site of laccases along with interacting residues. Left panel: plant laccase, right panel: fungal laccase, (A) monomer, (B) dimer, (C) trimer, (D) tetramer. Captions for supplementary videos: S Video 1. Movie of plant laccase complexed with monomer sinapyl alcohol during 25 ns MD


simulation. S Video 2. Movie of fungal laccase complexed with monomer sinapyl alcohol during 25 ns MD simulation. S Video 3. Movie of plant laccase complexed with tetramer guaiacyl β-O-4 syringyl β- β syringyl β-O-4 guaiacyl during 25 ns MD simulation. S Video 4. Movie of fungal laccase complexed with tetramer guaiacyl β-O-4 syringyl β- β syringyl β-O-4 guaiacyl during 25 ns MD simulation.

31

32


33


34


35


36


37


38


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

Plant Laccases

Fungal Laccases

A) Biochemical 1) pH optima range

7.0-10.0 (neutral to alkaline) (a, b)

2.0-6.5 (acidic) (c, d)

2) Isoelectric point

5.0-9.0 (e)

3.0-5.0 (f)

On an average >100 kDa (a, b)

On an average

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