NAD(P)H-dependent aldose reductase from the xylose-assimilating yeast Candida tenuis. Isolation, characterization and biochemical properties of the enzyme.
During growth on d-xylose the yeast Candida tenuis produces one aldose reductase that is active with both NADPH and NADH as coenzyme. This enzyme has been isolated by dye ligand and anion-exchange chromatography in yields of 76%. Aldose reductase consists ofa single 43 kDa polypeptide with an isoelectric point of 4.70. Initial velocity, product inhibition and binding studies are consistent with a compulsory-ordered, ternary-complex mechanism with coenzyme binding first and leaving last. The catalytic efficiency (kcat/Km) in d-xylose reduction at pH 7 is more than 60-fold higher than that in xylitol oxidation and reflects significant differences in the corresponding catalytic centre activities as well as apparent substrate-binding constants. The enzyme prefers NADP(H) approx. 2-fold to NAD(H), which is largely due to better apparent binding of the phosphorylated form of the coenzyme. NADP+ is a potent competitive inhibitor of the NADH-linked aldehyde reduction (Ki 1.5 microM), whereas NAD+ is not. Unlike mammalian aldose reductase, the enzyme from C. tenuis is not subject to oxidation-induced activation. Evidence of an essential lysine residue located in or near the coenzyme binding site has been obtained from chemical modification of aldose reductase with pyridoxal 5'-phosphate. The results are discussed in the context of a comparison of the enzymic properties of yeast and mammalian aldose reductase.
Neuhauser
W
W
Division of Biochemical Engineering, Institute of Food Technology, Universität für Bodenkultur Wien, Muthgasse 18, A-1190 Vienna, Austria.
Haltrich
D
D
Kulbe
K D
KD
Nidetzky
B
B
D000449:Aldehyde Reductase / Q000302:isolation & purification* / Q000378:metabolism*; D002175:Candida / Q000187:drug effects / Q000201:enzymology*; D007700:Kinetics; D009249:NADP / Q000378:metabolism*; D013379:Substrate Specificity; D014994:Xylose / Q000378:metabolism* / Q000494:pharmacology
D009249:NADP; D014994:Xylose; D000449:Aldehyde Reductase