Investigation of the Role of a Conserved Glycine Motif in the Saccharomyces cerevisiae Xylose Reductase

All yeast xylose reductases, with the exception of that from Schizosaccharomyces pombe, possess the catalytic and coenzyme-binding elements from both the aldo–keto reductase and short-chain dehydrogenase–reductase (SDR) enzyme families in their primary sequences. In the Saccharomyces cerevisiae xylose reductase (XR), the SDR-like coenzyme-binding GXXXGXG motif (Gly motif) is located between residues 128 and 134, with the third Gly residue being replaced by an Asp. We used site-directed mutagenesis to study the role of this SDR-like Gly motif in the S. cerevisiae xylose reductase. Site-directed mutagenesis of the individual conserved Gly residue positions (G128A, G132A, D134G, and D134A) did not significantly affect the specific activity, kinetic constants (K_m, K_cat, and K_cat/K_m), or dissociation constants (K_d) in any of the variants compared with the wild type. Deletion of the entire Gly motif produced an unstable protein that could not be purified. These results indicate that the SDR-like Gly motif likely provides support to the overall structure of the enzyme, but it does not contribute directly to coenzyme binding in this XR.