Structure of a serine-type glutathione S-transferase of Ceriporiopsis subvermispora and identification of the enzymatically important non-canonical residues by functional mutagenesis.

Abstract Ceriporiopsis subvermispora (C. subvermispora), one of the white-rot fungi, is known as a selective lignin degrader of the woody biomass. Glutathione S-transferases (GSTs) are multifunctional enzymes that are capable of catalyzing the reactions involved in detoxification and metabolic pathways. In this study, a GST of C. subvermispora , named CsGST63524, was overexpressed in E. coli , and then purified by affinity, anion exchange, and size exclusion column chromatography. The crystal structures of the CsGST63524 in ligand-free and complex with GSH were refined at 2.45 and 2.50 Å resolutions, respectively. The sulfur atom of glutathione forms a hydrogen bond with Ser21 of CsGST63524, indicating it is a serine-type GST. Mutagenesis of Ser21 unexpectedly indicated that this serine residue is not essential for the enzymatic activity of CsGST63524. Comparative sequence and structural analyses, together with functional mutagenesis, newly identified the enzymatically important non-canonical amino acid residues, Asn23 and Tyr45, other than the serine residue. Highlights • GSH-free and -bound structures of a C. subvermispora GST, CsGST63524, were solved. • Ser21 is located closest to a sulfur of GSH, indicating CsGST63524 is a serine-type. • S21A mutant unexpectedly indicated Ser21 is not crucial for the enzymatic activity. • Functional mutagenesis revealed Asn23 and Tyr45 are crucial for enzymatic activity. • A putative substrate-binding site was deduced from those of homologous proteins. [ABSTRACT FROM AUTHOR]