COMPUTATIONAL STUDIES ON LIP H ISOLATED FROM GANODERMA LUCIDUM GD 88

Ganoderma lucidum is a basidiomycete fungus that produces ligninase for the modification of lignin. Lignin peroxidase (LiP) is a glycoprotein that acts on the recalcitrant cell wall component lignin. In the present study, the phylogenetic analysis of Ganoderma lucidum GD88 with the partial coding sequence (cds) of other LiP isoforms was performed using MEGA6. After determination of the open reading frame, the +3 frame nucleotide sequence was converted to protein using the EMBOSS Transseq and the secondary structure was predicted using the Chou and Fasman Secondary Structure Prediction server (CFSSP). Protein modeling was also performed by SWISS-MODEL. The obtained result shows that the lipH partial cds of Ganoderma lucidum GD88 is homologous to the lipD gene of Phanerochaete chrysosporium. The secondary structure prediction result revealed that the percent content of the helix (67) is higher than the percent contents of sheet (53.4) and turns (13.6). According to the generated model, LiP H protein is a homodimer with chains A and B. The heme acts as a ligand and plays a major role in structure stabilization.


INTRODUCTION
Fungi are a very good source of lignin peroxidase, and the white rot fungi are the best-known producers of lignolytic enzymes (Kirk and Farrell, 1987), followed by brown and soft rot fungi (Niladevi, 2009).The most studied lignin-degrading system is that of Phanerochaete chrysosporium (Reddy and D'Souza, 1994;Cameron et al., 2000;Macarena et al., 2005).The other white rot fungi producing lignin peroxidase are Phlebia floridensis (Arora and Gill, 2004), and Panus tigrinus (Leontievsky et al., 1994).Among bacteria, the actinomycetes are potent producers of ligninolytic enzymes, and extracellular lignin peroxidase has been identified in different strains of Streptomyces, such as S. viridosporus, S. chromofuscus and S. psammoticus (Ramachandra et al., 1987;Pasti et al., 1990;Niladevi and Prema, 2005).Even though a large number of bacterial strains have been studied for lignin degradation, the production of lignin peroxidase is restricted to few strains of Pseudomonas (Yang et al., 2006).
Lignin peroxidase (EC 1.11.14) is a heme-containing lignin-modifying enzyme secreted by basidiomycete filamentous fungi and can degrade the recalcitrant cell wall component lignin.LiPs are oligomannose-type glycoproteins with a number of possible O-glycosylation sites and one or more Nglycosylation sites (Eriksson and Bermek, 2009).The structure of LiP has been elucidated by x-ray crystallography and other methods (Edwards et al., 1993;Choinowski et al., 1999).The formation, inactivation and conversion of lignin peroxidase to the native enzyme have also been revealed (Wariishi et al., 1989).
Ganoderma lucidum is an economically important basidiomycete because of its medicinal properties and role in traditional medicine of eastern countries.The name "lucidum" means shiny in Latin, referring to the varnished-like fruiting body of the mushroom.This annual mushroom propagates on a large variety of dead or dying trees, e.g., deciduous trees, especially oak, maple, elm, willow and magnolia (Wasser, 2005).However, its lignin-degrading profile has not been ad-equately studied and the structural characterization of the enzyme remains to be elucidated.In the present study we describe the structure of LiP H isolated from Ganoderma lucidum GD88 by prediction and modeling tools.

Source of sequence
Basidiomycete fungi were isolated from different locations of Kerala and maintained on potato dextrose agar.From the isolated strains, the maximum lignolytic enzyme producing strain, GD88, was identified to be Ganoderma lucidum by 18S ribotyping (Hariharan and Nambisan, 2013).The LiP produced by this strain was purified and characterized.The DNA from the strain was isolated and the lipH coding sequence amplified by PCR using the primer pair 5' GCAATTGCCATCTCGCCC and 3' ACAC-GGTTAATGAGCTGG (Janse et al., 1998).The amplified product was gel eluted and sequenced.The sequence obtained was deposited in the NCBI database (JQ040847.1).The partial coding sequences of other LiP isoforms were also accessed from the NCBI database.

Phylogenetic analysis and protein modeling
The nucleotide sequence (partial cds) of Ganoderma lucidum GD88 and the partial cds of other LiP isoforms available in the NCBI database are listed in Table 1.The sequences were aligned by CLuSTAL W2 and the phylogenetic analysis was done using the software MEGA 6 with 500 bootstrap replications.The open reading frame (ORF) of the nucleotide sequence of LiP H was determined using ORF finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html)and the corresponding reading frame was converted to protein sequence using the EMBOSS Transseq (http://www.ebi.ac.uk/Tools/st/ emboss_transeq/).The secondary structure of the converted LiP protein sequence was elucidated using the CFSSP algorithm (http://www.biogem.org/tool/chou-fasman/).Protein modeling was performed using the SWISS-MODEL (http://swissmodel.expasy.org/)tool.The SWISS-MODEL template library (SMTL version 2014-12-03) was searched with BLAST and HHBlits for evolutionary related structures matching the target sequence.Models were built based on the target-template alignment using Promod-II.Ligand modeling and the model quality was also estimated.

Phylogenetic analysis
The partial cDNA nucleotide sequences of the available LiP genes were aligned using CLuSTAL W2, and a phylogenetic tree was constructed using MEGA 6.This shows that the lipH of Ganoderma lucidum GD88 is homologous to the lipD gene of Phanerochaete chrysosporium (Fig. 1).
The reading frame coding for LiP H was found to be +3, and the corresponding nucleotide sequence was converted to protein sequence.The sequence is shown below: The obtained protein sequence was used for the further studies.

Secondary structure prediction
The secondary structure prediction tool CFSSP was used to determine the structure of the LiP H protein.The protein sequence generated was used as the template for the structure prediction.The sequence consists of 107 amino acids.The percentage of sheet (53.4) was found to be higher in the protein, followed by helix (67) and turns (13.6).Thus, the α-helices predominate over β-sheets and turns in LiP H protein molecule of Ganoderma lucidum GD 88; this supports the secondary structure of LiP described by Wong (2009).

Construction of a protein model
SWISS MODEL is an automated system for modeling the 3D structure of a protein using homology modeling.The protein sequence generated was used as the target sequence and the template selection was done.BLAST and HHBlits were used for the template search against the SWISS MODEL Template Library (SMTL).
The search deduced 184 templates from all the SMTL profiles and are listed in Table 2.The template's quality was predicted from features of the target-template alignment and 62 of them were found to be matching templates.These 62 templates were used for building the homology model for the target using Promod-II.
Determining the most accurate model is a crucial step in homology modeling.When combining the estimates of each property, the most likely structural similarity is the value at which the joint distribution is maximized, termed the global quality estimation score (GMQE) (Schwede et al., 2003).The accuracy and reliability of the modeled protein can be estimated from the Global Mean Quality Estimation (GMQE) score, which is achieved through the QMEAN Server method.Among the models predicted, the one with highest GMQE score was selected and the reliable model was found to be generated by catalase peroxidase protein as template.The model features are shown in Table 3.According to the model, LiP H is a homodimer with two domains: chain A and chain B that supports the LiP structure described by Wong (2009).It has protoporphyrin IX containing heme as the ligand, which is in contact with chain B through amino acid residues R35, S36, F39 and R40.In the monomer model generated, chain A is in contact with the heme through residues T33, R35, S36 and F39 (Fig. 3).
To conclude, the phylogenetic analysis of the nucleotide sequence (partial cds) of lipH from Ganoderma lucidum GD88 with the partial cds of other LiP iso- forms points to the identity of lipH with the lipD gene of Phanerochaete chrysosporium.The secondary structure prediction of LiP H showed more α helices compared to β-sheets and turns.The present study has generated the most reliable model of LiP H using catalase peroxidase protein as template.The ligand associated with LiP H was found to be protoporphyrin IX containing iron.

Fig. 3 .
Fig. 3. Residues in contact with the heme of LiP H. а -residues in monomer model; b -residues in homodimer model; c -monomer model; d -homodimer model.

Table 1 .
lip partial cds nucleotide sequences used for phylogenetic analysis.

Table 2 .
List of templates used for model building by SWISS MODEL.

Table 3 .
Description of the LiP H protein model generated by catalase peroxidase protein as template.