computational studies on lip h isolated ganoderma …

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Arch. Biol. Sci., Belgrade, 67(3), 817-828, 2015 DOI:10.2298/ABS141014041P

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), fol-lowed 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 peroxi-dase 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 peroxi-dase has been identified in different strains of Strep-tomyces, 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 per-oxidase is restricted to few strains of Pseudomonas (Yang et al., 2006).

Lignin peroxidase (EC 1.11.14) is a heme-con-taining lignin-modifying enzyme secreted by ba-sidiomycete 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 N-glycosylation sites (Eriksson and Bermek, 2009). The structure of LiP has been elucidated by x-ray crystal-lography and other methods (Edwards et al., 1993; Choinowski et al., 1999). The formation, inactivation and conversion of lignin peroxidase to the native en-zyme have also been revealed (Wariishi et al., 1989).

Enzymes from basidiomycete strains are ca-pable of decolorizing synthetic dyes (Gomes et al.,

COMPUTATIONAL STUDIES ON LIP H ISOLATED FROM GANODERMA LUCIDUM GD88

Nayana Parambayil*, Aiswarya Chenthamarakshan, Arinnia Anto, Sudha Hariharan and Padma Nambisan

Plant Biotechnology Laboratory, Department of Biotechnology, Cochin University of Science and Technology, Cochin 682022, Kerala, India.

*Corresponding author: [email protected]

Abstract: 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 phylo-genetic 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 sec-ondary 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.

Key words: Ganoderma lucidum; lignin peroxidase; MEGA 6; CFSSP; SWISS-MODEL

Received October 14, 2014; Revised December 15, 2014; Accepted December 17, 2014

818 Parambayil et al.

2009), reactive dyes (Vaithanomsat et al., 2010) and other industrial dyes (Lopez et al., 2006). In the food industry, lignin peroxidase has been used as a source of natural aromatics and in the production of vanillin (Lesage-Meessen et al., 1996; Lomascolo et al., 1999; Barbosa et al., 2008). It has been used for the decolorization of kraft pulp and mill efflu-ents in paper-pulp industries (Ferrer et al., 1991; Bajpai, 2004; Sigoillot et al., 2005). It can also carry out degradation of azo, heterocyclic, reactive and polymeric dyes, mineralization of environmental contaminants, xenobiotic and pesticides degrada-tion (Bumpus and Aust, 1987; Abraham et al., 2002; Ohtsubo et al., 2004; Robles-Hernández et al., 2008; Gomes et al., 2009; Wen et al., 2009). Ligninases thus find wide application in organic synthesis, medical, pharmaceutical, cosmetics and nanotechnology ap-plications (Maciel et al., 2010).

Ganoderma lucidum is an economically impor-tant 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 pres-ent study we describe the structure of LiP H isolated from Ganoderma lucidum GD88 by prediction and modeling tools.

MATERIALS AND METHODS

Source of sequence

Basidiomycete fungi were isolated from different locations of Kerala and maintained on potato dex-trose agar. From the isolated strains, the maximum lignolytic enzyme producing strain, GD88, was iden-tified 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 cod-ing sequence amplified by PCR using the primer pair 5’ GCAATTGCCATCTCGCCC and 3’ ACAC-GGTTAATGAGCTGG (Janse et al., 1998). The am-plified product was gel eluted and sequenced. The sequence obtained was deposited in the NCBI da-tabase (JQ040847.1). The partial coding sequences of other LiP isoforms were also accessed from the NCBI database.

Fig. 1. Phylogenetic distribution of LiP isoform partial cds.

STuDIES ON LIP H 819

Phylogenetic analysis and protein modeling

The nucleotide sequence (partial cds) of Gano-derma 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 boot-strap 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 us-ing the CFSSP algorithm (http://www.biogem.org/tool/chou-fasman/). Protein modeling was per-formed using the SWISS-MODEL (http://swissmo-del.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.

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

Sl. No. Species Genbank accession no. Gene

1. Phanerochaete chrysosporium strain 36210 Gu119913.1 Lignin peroxidase isozyme H8 (lipH8)

2. Phanerochaete chrysosporium clone pchl6 EF644562.1 Lignin peroxidase isoform D (lipD) gene

3. Phanerochaete chrysosporium clone pchl5 EF644561.1 Lignin peroxidase isoform A (lipA) gene

4. Phanerochaete chrysosporium clone pchl4 EF644560.1 Lignin peroxidase isoform G (lipG) gene

5. Phanerochaete chrysosporium clone pchl3 EF644559.1 Lignin peroxidase isoform B (lipB)

6. Phanerochaete chrysosporium clone pchl2 EF644558.1 Lignin peroxidase isoform E (lipE) gene

7. Phanerochaete chrysosporium clone pchl1 EF644557.1 Lignin peroxidase isoform J (lipJ)

8. Ganoderma lucidum strain GD88 JQ040847.1 Lignin peroxidase H (lipH)

9. Phlebia sp. b19 EF491859.1 Lignin peroxidase precursor (lip4)



Fig. 2. Secondary structure prediction of LiP H from Ganoderma lucidum GD88 showing predominance of α helices.


RESULTS AND DISCUSSION

Phylogenetic analysis

The partial cDNA nucleotide sequences of the avail-able 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 Phanero-chaete chrysosporium (Fig. 1).

DNA to protein conversion

The nucleotide sequence (partial cds) of Gano-derma lucidum GD88 (JQ040847.1), downloaded

in FASTA format from the NCBI site (http://www.ncbi.nlm.nih.gov/), was loaded into ORF Finder. 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:

NCHLAPRIDEERSEMR*VM*IAEATRPAAPGSM-CTLRSLVFRGACLFE*AKGKFGGGGGADGSIMIFDTIETAFHPNIGLDEVVALQNGRLLLNALAWFLASSLTVX

The obtained protein sequence was used for the further studies.

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.


Secondary structure prediction

The secondary structure prediction tool CFSSP was used to determine the structure of the LiP H pro-tein. 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 pre-dominate 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 model-ing. The protein sequence generated was used as the target sequence and the template selection was done.

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

Sl. No. Template Oligo-state Coverage Description

1. 1ub2.1.A homo-dimer 0.83 Catalase-peroxidase

2. 2fxg.1.A homo-dimer 0.83 catalase-peroxidase protein

3. 2vka.1.A monomer 0.77 VERSATILE PEROXIDASE VPL2

4. 1lyk.1.A monomer 0.77 Peroxidase

5. 1h3j.1.A monomer 0.77 PEROXIDASE

6. 2w23.1.A monomer 0.76 VERSATILE PEROXIDASE VPL2

7. 4fcs.1.A monomer 0.75 Versatile peroxidase VPL2

8. 3wnu.1.A homo-dimer 0.8 Catalase-peroxidase

9. 1sj2.1.A homo-dimer 0.8 Peroxidase/catalase T

10. 2cca.1.A homo-dimer 0.8 PEROXIDASE/CATALASE T

11. 4c50.1.A homo-dimer 0.8 CATALASE-PEROXIDASE

12. 2ccd.1.A homo-dimer 0.8 PEROXIDASE/CATALASE T


14. 3vlk.1.A homo-dimer 0.79 Catalase-peroxidase 2

15. 3vll.1.B homo-dimer 0.79 Catalase-peroxidase 2

16. 3uw8.1.A homo-dimer 0.79 Catalase-peroxidase 2

17. 3vlh.1.A homo-dimer 0.79 Catalase-peroxidase 2

18. 4ka6.1.A homo-dimer 0.8 Catalase-peroxidase

19. 2dv1.1.A homo-dimer 0.79 Peroxidase/catalase

20. 3n3s.1.A homo-dimer 0.79 Catalase-peroxidase

21. 3n3q.1.A homo-dimer 0.79 Catalase-peroxidase

22. 1x7u.1.A homo-dimer 0.79 catalase-peroxidase protein KatG

23. 3ut2.1.A homo-dimer 0.79 Catalase-peroxidase 2

24. 3n3r.1.A homo-dimer 0.79 Catalase-peroxidase

25. 2fxj.1.A homo-dimer 0.79 catalase-peroxidase protein




27. 1mwv.1.A homo-dimer 0.79 catalase-peroxidase protein KatG

28. 2v23.1.A monomer 0.79 CYTOCHROME C PEROXIDASE

29. 1a2f.1.A monomer 0.79 CYTOCHROME C PEROXIDASE

30. 1cpg.1.A monomer 0.79 CYTOCHROME C PEROXIDASE

31. 7ccp.1.A monomer 0.79 CYTOCHROME C PEROXIDASE

32. 1dj5.1.A monomer 0.79 CYTOCHROME C PEROXIDASE

33. 1ccg.1.A monomer 0.79 CYTOCHROME C PEROXIDASE

34. 1ccj.1.A monomer 0.79 CYTOCHROME C PEROXIDASE

35. 1cci.1.A monomer 0.79 CYTOCHROME C PEROXIDASE

36. 1dso.1.A monomer 0.79 CYTOCHROME C PEROXIDASE

37. 1bva.1.A monomer 0.79 PROTEIN (CYTOCHROME C PEROXIDASE)

38. 4a78.1.A monomer 0.78 CYTOCHROME C PEROXIDASE, MITOCHONDRIAL

39. 2x08.1.A monomer 0.78 CYTOCHROME C PEROXIDASE, MITOCHONDRIAL

40. 4a71.1.A monomer 0.78 CYTOCHROME C PEROXIDASE, MITOCHONDRIAL

41. 3vlm.1.A homo-dimer 0.75 Catalase-peroxidase 2

42. 1itk.1.A homo-dimer 0.75 catalase-peroxidase

43. 2xj5.1.A monomer 0.78 CYTOCHROME C PEROXIDASE, MITOCHONDRIAL

44. 1bek.1.A monomer 0.78 YEAST CYTOCHROME C PEROXIDASE


46. 1kxm.1.A monomer 0.78 Cytochrome c Peroxidase

47. 2jti.1.A hetero-oligomer 0.78 Cytochrome c peroxidase, mitochondrial

48. 1cca.1.A monomer 0.78 CYTOCHROME C PEROXIDASE

49. 3e2o.1.A monomer 0.78 Cytochrome c peroxidase

50. 2b12.1.A hetero-oligomer 0.78 Cytochrome c peroxidase, mitochondrial

51. 4cvi.1.A monomer 0.78 CYTOCHROME C PEROXIDASE, MITOCHONDRIAL

52. 2b11.1.A hetero-oligomer 0.78 Cytochrome c peroxidase, mitochondrial

53. 2b0z.1.A hetero-oligomer 0.78 Cytochrome c peroxidase, mitochondrial

54. 3m2c.1.A monomer 0.78 Cytochrome c peroxidase, mitochondrial

55. 4jb4.1.A monomer 0.78 Cytochrome c peroxidase, mitochondrial

56. 1z53.1.A monomer 0.78 Cytochrome c peroxidase, mitochondrial

57. 4nfg.1.A hetero-oligomer 0.78 Cytochrome c peroxidase, mitochondrial


59. 1a2g.1.A monomer 0.78 CYTOCHROME C PEROXIDASE

60. 1bem.1.A monomer 0.78 CYTOCHROME C PEROXIDASE

61. 2rc2.1.A monomer 0.78 Cytochrome C Peroxidase

62. 1bej.1.A monomer 0.78 CYTOCHROME C PEROXIDASE

Table 2 continued:



63. 3ccx.1.A monomer 0.78 CYTOCHROME C PEROXIDASE

64. 1ccl.1.A monomer 0.78 CYTOCHROME C PEROXIDASE

65. 3exb.1.A monomer 0.78 Cytochrome c peroxidase

66. 2cep.1.A monomer 0.78 CYTOCHROME C PEROXIDASE

67. 1bep.1.A monomer 0.78 YEAST CYTOCHROME C PEROXIDASE

68. 1cpf.1.A monomer 0.78 CYTOCHROME C PEROXIDASE

69. 1dcc.1.A monomer 0.78 CYTOCHROME C PEROXIDASE

70. 1ccp.1.A monomer 0.78 YEAST CYTOCHROME C PEROXIDASE

71. 2anz.1.A monomer 0.78 Cytochrome c peroxidase, mitochondrial

72. 1ccb.1.A monomer 0.78 CYTOCHROME C PEROXIDASE

73. 1mk8.1.A monomer 0.78 Cytochrome c Peroxidase

74. 1mkq.1.A monomer 0.78 Cytochrome c Peroxidase

75. 4a7m.1.A monomer 0.78 CYTOCHROME C PEROXIDASE, MITOCHONDRIAL



78. 1dse.1.A monomer 0.78 CYTOCHROME C PEROXIDASE

79. 2v2e.1.A monomer 0.77 CYTOCHROME C PEROXIDASE

80. 2xil.1.A monomer 0.77 CYTOCHROME C PEROXIDASE, MITOCHONDRIAL

81. 1ebe.1.A monomer 0.77 CYTOCHROME C PEROXIDASE

82. 1apx.1.A homo-dimer 0.7 CYTOSOLIC ASCORBATE PEROXIDASE

83. 4ccx.1.A monomer 0.77 CYTOCHROME C PEROXIDASE

84. 1beq.1.A monomer 0.77 CYTOCHROME C PEROXIDASE

85. 1itk.1.A homo-dimer 0.73 catalase-peroxidase

86. 3r99.1.A monomer 0.77 Cytochrome c peroxidase

87. 1stq.1.A monomer 0.77 Cytochrome c peroxidase, mitochondrial

88. 1jci.1.A monomer 0.77 Cytochrome C Peroxidase

89. 1sog.1.A monomer 0.77 Cytochrome c peroxidase

90. 1jdr.1.A monomer 0.77 Cytochrome c Peroxidase

91. 1s6v.1.A hetero-oligomer 0.77 Cytochrome c peroxidase, mitochondrial

92. 1llp.1.A monomer 0.59 LIGNIN PEROXIDASE

93. 3vlh.1.A homo-dimer 0.73 Catalase-peroxidase 2

94. 1cyf.1.A monomer 0.77 CYTOCHROME C PEROXIDASE

95. 1kxn.1.A monomer 0.77 cytochrome c peroxidase

96. 2icv.1.A monomer 0.77 Cytochrome c peroxidase, mitochondrial

97. 1ccc.1.A monomer 0.77 CYTOCHROME C PEROXIDASE

98. 1cck.1.A monomer 0.77 CYTOCHROME C PEROXIDASE

Table 2 continued:




100. 2cl4.1.A monomer 0.7 ASCORBATE PEROXIDASE

101. 1iyn.1.A monomer 0.73 Chloroplastic ascorbate peroxidase

102. 1cmu.1.A monomer 0.76 CYTOCHROME C PEROXIDASE

103. 2as2.1.A monomer 0.76 Cytochrome c peroxidase, mitochondrial

104. 1b80.1.A monomer 0.59 PROTEIN (RECOMBINANT LIGNIN PEROXIDASE H8)

105. 1b85.1.A monomer 0.59 Ligninase H8

106. 3vlk.1.A homo-dimer 0.72 Catalase-peroxidase 2

107. 3uw8.1.A homo-dimer 0.72 Catalase-peroxidase 2

108. 3vlm.1.A homo-dimer 0.72 Catalase-peroxidase 2

109. 3vll.1.B homo-dimer 0.72 Catalase-peroxidase 2

110. 3rrw.1.A monomer 0.72 Thylakoid lumenal 29 kDa protein, chloroplastic

111. 1krj.1.A monomer 0.75 Cytochrome c Peroxidase

112. 1qpa.1.A homo-dimer 0.59 LIGNIN PEROXIDASE

113. 2y6a.1.A homo-dimer 0.68 ASCORBATE PEROXIDASE

114. 2y6b.1.A homo-dimer 0.68 ASCORBATE PEROXIDASE

115. 2xif.1.A monomer 0.68 ASCORBATE PEROXIDASE

116. 2vcs.1.A monomer 0.68 ASCORBATE PEROXIDASE

117. 3n3r.1.A homo-dimer 0.71 Catalase-peroxidase

118. 2fxg.1.A homo-dimer 0.71 catalase-peroxidase protein


120. 3n3q.1.A homo-dimer 0.71 Catalase-peroxidase

121. 3n3s.1.A homo-dimer 0.71 Catalase-peroxidase

122. 2fxj.1.A homo-dimer 0.71 catalase-peroxidase protein

123. 1x7u.1.A homo-dimer 0.71 catalase-peroxidase protein KatG


125. 3zcy.1.A monomer 0.68 ASCORBATE PEROXIDASE

126. 1sj2.1.A homo-dimer 0.71 Peroxidase/catalase T


128. 2cca.1.A homo-dimer 0.71 PEROXIDASE/CATALASE T

129. 2wd4.1.A monomer 0.68 ASCORBATE PEROXIDASE

130. 1u2k.1.A monomer 0.71 Peroxidase/catalase HPI

131. 1u2l.1.A monomer 0.71 Peroxidase/catalase HPI

132. 4ged.1.A hetero-oligomer 0.7 Ascorbate peroxidase

133. 1v0h.1.A monomer 0.67 ASCORBATE PEROXIDASE

134. 3riv.1.A monomer 0.7 Ascorbate peroxidase

Table 2 continued:



135. 3e2n.1.A monomer 0.71 Cytochrome c peroxidase

136. 1mwv.1.A homo-dimer 0.7 catalase-peroxidase protein KatG


138. 3ut2.1.A homo-dimer 0.72 Catalase-peroxidase 2


140. 2ccd.1.A homo-dimer 0.7 PEROXIDASE/CATALASE T

141. 3riw.1.A monomer 0.69 Ascorbate peroxidase

142. 1u2j.6.A monomer 0.7 Peroxidase/catalase HPI

143. 1u2j.1.A monomer 0.7 Peroxidase/catalase HPI

144. 3zch.1.A homo-dimer 0.66 ASCORBATE PEROXIDASE

145. 3zcg.1.A homo-dimer 0.66 ASCORBATE PEROXIDASE


147. 4bm1.1.A monomer 0.59 MANGANESE PEROXIDASE 4

148. 3q3u.1.A monomer 0.59 Lignin peroxidase

149. 3fm6.1.A monomer 0.58 Versatile peroxidase VPL2


151. 3fmu.1.A monomer 0.58 Versatile peroxidase VPL2

152. 1lyc.1.A monomer 0.59 Peroxidase

153. 3fjw.1.A monomer 0.57 Versatile peroxidase VPL2

154. 3fkg.1.A monomer 0.57 Versatile peroxidase VPL2


156. 4blk.1.A monomer 0.57 VERSATILE PEROXIDASE I

157. 2boq.1.A monomer 0.57 VERSATILE PEROXIDASE VPL2

158. 1mnp.1.A monomer 0.6 MANGANESE PEROXIDASE

159. 1qgj.1.A monomer 0.64 PEROXIDASE N

160. 1arv.1.A monomer 0.58 PEROXIDASE

161. 4fdq.1.A monomer 0.56 Versatile peroxidase VPL2

162. 1ly8.1.A monomer 0.58 Peroxidase

163. 1mn1.1.A monomer 0.59 MANGANESE PEROXIDASE

164. 1mn2.1.A monomer 0.59 MANGANESE PEROXIDASE

165. 4fef.1.A monomer 0.55 Versatile peroxidase VPL2

166. 4fcn.1.A monomer 0.55 Versatile peroxidase VPL2

167. 4g05.1.A monomer 0.55 Versatile peroxidase VPL2

168. 2ylj.1.A monomer 0.63 PEROXIDASE C1A

169. 1gwo.1.A monomer 0.62 PEROXIDASE C1A

170. 3wnu.1.A homo-dimer 0.56 Catalase-peroxidase

Table 2 continued:



171. 1ub2.1.A homo-dimer 0.54 Catalase-peroxidase

172. 1llp.1.A monomer 0.24 LIGNIN PEROXIDASE

173. 1b80.1.A monomer 0.24 PROTEIN (RECOMBINANT LIGNIN PEROXIDASE H8)

174. 1b85.1.A monomer 0.24 Ligninase H8

175. 1qpa.1.A homo-dimer 0.24 LIGNIN PEROXIDASE

176. 3zwl.1.A hetero-oligomer 0.33 EuKARYOTIC TRANSLATION INITIATION FACTOR 3 SuBuNIT I

177. 4czy.1.A hetero-oligomer 0.34 PAB-DEPENDENT POLY(A)-SPECIFIC RIBONuCLEASE SuBuNIT PAN2

178. 2j04.1.A hetero-oligomer 0.25 HYPOTHETICAL PROTEIN YPL007C

179. 3fm0.1.A monomer 0.23 Protein CIAO1

180. 4mk0.1.B hetero-oligomer 0.23 Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1

181. 1a0r.1.A hetero-oligomer 0.23 TRANSDuCIN (BETA SuBuNIT)

182. 2bcj.1.B hetero-oligomer 0.23 Guanine nucleotide-binding protein G(I)/G(S)/G(T) beta subunit 1

183. 4czv.1.A monomer 0.24 PAB-DEPENDENT POLY(A)-SPECIFIC RIBONuCLEASE SuBuNIT PAN2

184. 1xhj.1.A monomer 0.16 Nitrogen Fixation Protein Nifu

185. 2jnv.1.A monomer 0.16 Nifu-like protein 1, chloroplast

186. 1th5.1.A monomer 0.15 Nifu1

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

Sl. No. Template Oligo-State Seq Identity Seq Similarity GMQE Ligand

1. 2fxg.1.A Homo-dimer 18.82 0.30 0.48 Protoporphyrin IX containing Fe

2. 2fxg.1.A Monomer 18.82 0.30 0.49 Protoporphyrin IX containing Fe

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 qual-ity 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 Glob-al 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 gen-erated 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 mono-mer 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 nucle-otide sequence (partial cds) of lipH from Ganoderma lucidum GD88 with the partial cds of other LiP iso-

Table 2 continued:


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.

Acknowledgments: The authors are grateful for the finan-cial support from the Cochin university of Science and Technology (Kerala, India), and the Kerala State Council for Science Technology and Environment (Kerala, India).

Authors’ contributions: All of the authors contributed equally in data acquisition, analysis, interpretation, and drafting of the article.

Conflict of interest disclosure: There is no conflict of in-terest between the authors.

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computational studies on lip h isolated ganoderma …

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