Sequence and Bioinformatic Analysis of Family 1 Glycoside Hydrolase (GH) 1 Gene from the Oomycete Pythium myriotylum Drechsler

• Published in: Applied biochemistry and biotechnology 2015-06, Vol.176 (3), p.772-781
• Main Authors: Nair, R. Aswati, Geethu, C, Sangwan, Amit, Pillai, P. Padmesh
• Format: Article
• Language: English
• Subjects: active sites; Amino Acid Sequence; amino acid sequences; Amino acids; binding sites; Biochemistry; Bioinformatics; Biotechnology; catalysts; cellulase; cellulases; Chemistry; Chemistry and Materials Science; Cloning; Cloning, Molecular; Computational Biology; Enzymes; genes; Glycoside Hydrolases - chemistry; Glycoside Hydrolases - genetics; glycosides; Models, Molecular; Molecular Sequence Data; molecular weight; nucleotide sequences; nutrition; Oomycetes; Pathogenesis; Phylogenetics; Phylogeny; plant pathogens; polymerase chain reaction; Protein Structure, Tertiary; Pythium - enzymology; Pythium - genetics; Pythium myriotylum; Sequence Alignment; Sequence Analysis; Sequence Homology, Nucleic Acid
• ISSN: 0273-2289; 1559-0291
• DOI: 10.1007/s12010-015-1610-6

The oomycetous phytopathogen Pythium myriotylum secretes cellulases for growth/nutrition of the necrotroph. Cellulases are multi-enzyme system classified into different glycoside hydrolase (GH) families. The present study deals with identification and characterization of GH gene sequence from P. myriotylum by a PCR strategy using consensus primers. Cloning of the full-length gene sequence using genome walker strategy resulted in identification of 1230-bp P. myriotylum GH gene sequence, designated as PmGH1. Analysis revealed that PmGH1 encodes a predicted cytoplasmic 421 amino acid protein with an apparent molecular weight of 46.77 kDa and a theoretical pI of 8.11. Tertiary structure of the deduced amino acid sequence showed typical (α/β)₈ barrel folding of family 1 GHs. Sequence characterization of PmGH1 identified the conserved active site residues, viz., Glu 181 and Glu 399, that function as acid-base catalyst and catalytically active nucleophile, respectively. Binding sites for N-acetyl-D-glucosamine (NAG) were revealed in the PmGH1 3D structure with Glu181 and Glu399 positioned on either side to form a catalytic pair. Phylogenetic analysis indicated a closer affiliation of PmGH1 with sequences of GH1 family. Results presented are first attempts providing novel insights into the evolutionary and functional perspectives of the identified P. myriotylum GH.