Purification and characterization of the 2-ketoaldonate reductase from Brevibacterium ketosoreductum ATCC21914.

2-Ketoaldonate reductase, which is involved in ketogluconate catabolism, was purified to homogeneity from Brevibacterium ketosoreductum ATCC21914. The enzyme was found to catalyze the reduction of 2,5-diketo-D-gluconate to 5-keto-D-gluconate, and to a lesser extent, 2-keto-D-gluconate to D-gluconate, and 2-keto-L-gluconate to L-idonate. The molecular mass of the reductase was 35 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 72 kDa by gel filtration, indicating that the native enzyme may exist as a dimer. The reductase was optimally active at pH 6.0 with NADPH as a preferred electron donor. The pI of 4.7 was measured for the enzyme. The apparent Km for 2,5-diketo-D-gluconate and NADPH were 5 microM and 10 microM, respectively. The amino-terminal amino acid sequence was NH2-Ala-Ser-Ile-Ser-Val-Ser-Val-Pro-Ser-Ala- Arg-Leu-Ala-Glu-Asp-Leu-Ser-Asp-Ile-Glu.