Purification and Characterization of the 2-Ketoaldonate Reductase from Brevibacterirnm ketosoreductum ATCC21914

Abstract

  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-gulonate 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 μM and 10 μM, 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.