Structure-function studies of xylose reductase from the yeast Saccharomyces cerevisiae.

摘要

Xylose reductase from Saccharomyces cerevisiae (SCXR) catalyzes the NAD(P)H-dependent reduction of xylose to xylitol. The role of the conserved GXXXGXD SDR (short chain dehydrogenase/reductase) cofactor binding motif in SCXR was investigated using site-directed mutagenesis. Individual substitution of Gly and Asp to Ala did not cause significant changes in K m, kcat, (kcat/Km), Kd or secondary structure in any of the variants compared to the wild type. These results indicate that the Gly motif does not directly participate in cofactor binding in SCXR.; The role of the conserved AKR (aldo-keto reductase) catalytic residues Asp44 and His111 in SCXR were investigated using site-directed mutagenesis. Asp44Ala mutant exhibited higher Km and lower kcat for xylose compared to the wild type, while the His111Ala mutant had no detectable activity. Mutant and wild-type SCXRs had similar Kd NADPH and secondary structure content. These results suggest that Asp44 and His111 are catalytically essential to SCXR.