2,5-Diketo-D-gluconate reductase, a novel enzyme that catalyzes the stereospecific NADPH-dependent reduction of 2,5-diketo-D-gluconate to 2-keto-L-gulonate, has been purified to homogeneity by sequential anion exchange, Cibacron blue F3GA affinity, and gel permeation chromatography from Corynebacterium sp. ATCC 31090. Molecular weight of the native form, determined by gel permeation chromatography, is 35,000 +/- 2,000. The subunit molecular weight, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is 34,000; thus, the enzyme is active as a monomer. A pI value of 4.4 is measured for the enzyme. Amino- and carboxyl-terminal sequences are consistent with that predicted by the DNA sequence of the reductase gene. At 25 degrees C, pH 6.4, the turnover number is 500 min-1, and the apparent Km values for 2,5-diketo-D-gluconate and NADPH are 26 mM and 10 microM, respectively. The enzyme is specific for NADPH, but the sugar binding site will also accept 5-keto-D-fructose and dihydroxyacetone as substrates. The enzyme is active over a broad pH range (pH 5-8) for the reduction of 2,5-diketo-D-gluconate; a sharp optimum at pH 9.2 is observed for the oxidation of 2-keto-L-gulonate. A Keq value of 5.6 X 10(-13) M indicates that reduction of substrate by NADPH is highly preferred. An activation energy of 12.3 kcal mol-1 is measured. Enzyme turnover is slow relative to dehydration of the gem-diol at C-5 of the substrate.