Identification of the yqhE and yafB genes encoding two 2,5-diketo-D-gluconate reductases in Escherichia coli

The identification of a gene (yiaE) encoding 2-ketoaldonate reductase (2KR) in our previous work led to the hypothesis that Escherichia coli has other ketogluconate reductases including 2,5-diketo-D-gluconate reductase (25DKG R) and to study of the related ketogluconate metabolism. By using the deduc ed amino acid sequences of 5-diketo-D-gluconate reductase (5KDGR) of Glucon obacter oxydans and 25DKGR of Corynebacterium sp., protein databases were s creened to detect homologous proteins. Among the proteins of E. coli, an ox idoreductase encoded by yjgU and having 56% similarity to 5KDGR of G. oxyda ns and two hypothetical oxidoreductases encoded by yqhE and yafB and having 49.8 and 42% similarity, respectively, to 25DKGR of Corynebacterium sp. we re detected. Recently, the yjgU gene aas identified as encoding 5KDGR and r enamed idnO (C. Bausch, N. Peekhaus, C. Utz, T. Blais, E. Murray, T. Lowary , and T. Conway, J. Bacteriol. 180:3704-3710, 1998). The pathways involved in the metabolism of ketogluconate by E. coli have been predicted by bioche mical analysis of purified enzymes and chemical analysis of the pathway int ermediates. The gene products of yqhE and yafB were identified as 25DKGR-A, and 25DKGR-B, respectively, catalyzing the reduction of 25KDG to 2-keto-L- gulonate (2KLG). The native 25DKGR-A, 25DKGR-B, and 5KDGR had apparent mole cular weights of about 30,000, 30,000, and 54,000, respectively. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels, all three enzymes showed protein bands with a molecular weight of about 29,000, which indicat ed that 25DKGR-A, 25DKGR-B, and 5KDGR may exist as monomeric, monomeric, an d dimeric proteins, respectively. The optimum pHs for reduction were 7.5, 7 .0, and 8.0, respectively. The 5KDGR was active with NADH, whereas 25DKGR-A and 25DKGR-B were active with NADPH as a preferred electron donor. 25DKG c an be converted to 5KDG by 2KR, which is then reduced to D-gluconate by 5KD GR. The pathways were compared with those of Erwinia sp. and Corynebacteriu m sp. A BLAST search of published and incomplete microbial genome sequences revealed that the ketogluconate reductases and their related metabolism ma y be widespread in many species.