Identification of lysine-78 as an essential residue in the Saccharomyces cerevisiae xylose reductase.
Yeast xylose reductases are hypothesized as hybrid enzymes as their primary sequences contain elements of both the aldo-keto reductases (AKR) and short chain dehydrogenase/reductase (SDR) enzyme families. During catalysis by members of both enzyme families, an essential Lys residue H-bonds to a Tyr residue that donates proton to the aldehyde substrate. In the Saccharomyces cerevisiae xylose reductase, Tyr49 has been identified as the proton donor. However, the primary sequence of the enzyme contains two Lys residues, Lys53 and Lys78, corresponding to the conserved motifs for SDR and AKR enzyme families, respectively, that may H-bond to Tyr49. We used site-directed mutagenesis to substitute each of these Lys residues with Met. The activity of the K53M variant was slightly decreased as compared to the wild-type, while that of the K78M variant was negligible. The results suggest that Lys78 is the essential residue that H-bonds to Tyr49 during catalysis and indicate that the active site residues of yeast xylose reductases match those of the AKR, rather than SDR, enzymes. Intrinsic enzyme fluorescence spectroscopic analysis suggests that Lys78 may also contribute to the efficient binding of NADPH to the enzyme.
Jeong
Eun Ye
EY
Department of Environmental Biology, University of Guelph, ON, Canada N1G 2W1.
Kim
In Seon
IS
Lee
Hung
H
D000449:Aldehyde Reductase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D020134:Catalytic Domain; D004789:Enzyme Activation; D005285:Fermentation; D008239:Lysine / Q000235:genetics; D008969:Molecular Sequence Data; D009249:NADP / Q000378:metabolism; D010427:Pentose Phosphate Pathway; D011485:Protein Binding; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics*
D011994:Recombinant Proteins; D009249:NADP; D000449:Aldehyde Reductase; D008239:Lysine