Yeast glutathione (GSH) reductase Glr1 is a dimeric flavo-oxidoreductase involved in cytoplasmic and mitochondrial redox regulatory systems. It reduces the oxidized GSH GSSG to the reduced form, GSH with NADPH as electron donor and FAD as coenzyme. Crystal structures and enzymatic mechanisms of GSH reductases from Escherichia coli and Homo sapiens have been well investigated, whereas the structural properties of yeast Glr1 remain unknown. Herein, we overexpressed Saccharomyces cerevisiae Glr1 in Pichia pastoris GS115 and determined its crystal structure at 2.40 A resolution. Although the overall structure and the active site are much conserved, obvious variety was found at the interface of Glr1 monomers when superimposed against the homolog from E. coli or human. The nonconserved C239 is exposed to the solvent and accessible to GSH or GSSG enriched in a microenvironment around the Glr1 molecules, leading to the partial and transient glutathionylation, as primarily identified from the 2Fo-Fc electron density map and further confirmed by biochemical assays. Meanwhile N278 at the vicinity of NADP-binding pocket was artificially glycosylated when heterogeneously overexpressed in P. pastoris. The highly motile oligosaccharide chain linked to N278 of the recombinant Glr1 interferes with the entry of NADPH, which results in a dramatic increase of Km for NAPDH and a significant decrease of turnover number, when compared with the native protein.
2007 Wiley-Liss, Inc.