Isolation and characterization of hydroxyl-radical-producing glycopeptide genes from the white-rot basidiomycete Phanerochaete chrysosporium
H Tanaka, G Yoshida, Y Baba, K Matsumura, S Itakura, A Enoki
During wood decay, the white-rot basidiomycete Phanerochaete chrysosporium secretes low-molecular-weight glycopeptides that catalyze a redox reaction between O2 and electron donors to produce hydroxyl radical. This reaction accounts for most of the hydroxyl radical produced in wood-degrading cultures of P. chrysosporium. In combination with phenol oxidases, hydroxyl radical is believed to play a role in lignin degradation. Low-molecular-weight fractions with high one-electron oxidation activity were isolated from wood-containing cultures of P. chrysosporium. They were composed of protein (55% w/w), neutral carbohydrate (25% w/w), and Fe(II) (0.04% w/w). A partially-purified glycopeptide was separated by tricine-SDS-PAGE and the bands were excised and digested with endoproteinase Asp-N. The N-terminal and four internal fragments were sequenced. BLAST and FASTA searches revealed no homologies between these amino-acid sequences and known proteins. However 70-100% similarities were found when the P. chrysosporium genome database was searched for the amino-acid sequences. cDNAs and two putative genes encoding these glycopeptides, glp1 and glp2, were isolated and sequenced. The 875-bp glp1 and 864-bp glp2 are located on scaffold 2 of the P. chrysosporium genome. These presumptive genes each consist of seven introns and eight exons. The latter encode a predicted mature peptide of 138 amino acids and a 22-amino-acid signal sequence for secretion. The predicted peptide sequences are nearly identical to N-terminal and internal sequences obtained from the partially-purified glycopeptide. The molecular weights of the deduced mature proteins coincide with the molecular weight of the glycopeptide as determined by tricine-SDS-PAGE. Finally, the amino-acid composition of the glycopeptide is nearly identical to the amino-acid compositions deduced from the glp1 and glp2 sequences. It appears that glp1 and glp2 encode the partially-purified hydroxyl-radical-producing glycopeptide Glp.
Keywords: wood decay, white rot, hydroxyl radical, glycopeptide