PURIFICATION AND PARTIAL CHARACTERIZATION OF AN ALDO-KETO REDUCTASE FROM SACCHAROMYCES-CEREVISIAE

A cytosolic aldo-keto reductase was purified from Saccharomyces cerevisae ATCC 26602 to homogeneity by affinity chromatography, chromatofocusing, and hydroxylapatite chromatography. The relative molecular weights of the aldo-keto reductase as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and size exclusion chromatography were 36,800 and 35,000, respectively, indicating that the enzyme is monomeric. Amino acid composition and N-terminal sequence analysis revealed that the enzyme is closely related to the aldose reductases of xylose-fermenting yeasts and mammalian tissues. The enzyme was apparently immunologically unrelated to the aldose reductases of other xylose-fermenting yeasts. The aldo keto reductase is NADPH specific and catalyzes the reduction of a variety of aldehydes. The best substrate for the enzyme is the aromatic aldehyde p-nitrobenzaldehyde (K-m = 46 mu M; k(cat)/K-m = 52,100 s(-1) M(-1)), whereas among the aldoses, DL-glyceraldehyde was the preferred substrate (K-m = 1.14 mM; k(cat)/K-m = 1,790 s(-1) M(-1)). The enzyme failed to catalyze the reduction of menadione and p-benzoquinone, substrates for carbonyl reductase. The enzyme was inhibited only slightly by 2 mM sodium valproate and was activated by pyridoxal 5'-phosphate. The optimum pH of the enzyme is 5. These data indicate that the S. cerevisiae aldo keto reductase is a monomeric NADPH-specific reductase with strong similarities to the aldose reductases.