Publications

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

  Yum D.-Y., Lee B.-Y., Pan J.-G.

  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 (25DKGR) and to study of the related ketogluconate metabolism. By using the de ... >> More

  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 (25DKGR) and to study of the related ketogluconate metabolism. By using the deduced amino acid sequences of 5-diketo-D-gluconate reductase (5KDGR) of Gluconobacter oxydans and 25DKGR of Corynebacterium sp., protein databases were screened to detect homologous proteins. Among the proteins of E. coli, an oxidoreductase encoded by yjgU and having 56% similarity to 5KDGR of G. oxydans and two hypothetical oxidoreductases encoded by yqhE and yafB and having 49.8 and 42% similarity, respectively, to 25DKGR of Corynebacterium sp. were detected. Recently, the yjgU gene was identified as encoding 5KDGR and renamed 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 biochemical analysis of purified enzymes and chemical analysis of the pathway intermediates. 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 molecular 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 indicated that 25DKGR-A, 25DKGR-B, and 5KDGR may exist as monomeric, monomeric, and 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 can be converted to 5KDG by 2KR, which is then reduced to D-gluconate by 5KDGR. The pathways were compared with those of Erwinia sp. and Corynebacterium sp. A BLAST search of published and incomplete microbial genome sequences revealed that the ketogluconate reductases and their related metabolism may be widespread in many species. << Less

  Appl. Environ. Microbiol. 65:3341-3346(1999) [PubMed] [EuropePMC]

• Crystal structure of 2,5-diketo-D-gluconic acid reductase A complexed with NADPH at 2.1-A resolution.

  Khurana S., Powers D.B., Anderson S., Blaber M.

  The three-dimensional structure of Corynebacterium 2, 5-diketo-D-gluconic acid reductase A (2,5-DKGR A; EC 1.1.1.-), in complex with cofactor NADPH, has been solved by using x-ray crystallographic data to 2.1-A resolution. This enzyme catalyzes stereospecific reduction of 2,5-diketo-D-gluconate (2 ... >> More

  The three-dimensional structure of Corynebacterium 2, 5-diketo-D-gluconic acid reductase A (2,5-DKGR A; EC 1.1.1.-), in complex with cofactor NADPH, has been solved by using x-ray crystallographic data to 2.1-A resolution. This enzyme catalyzes stereospecific reduction of 2,5-diketo-D-gluconate (2,5-DKG) to 2-keto-L-gulonate. Thus the three-dimensional structure has now been solved for a prokaryotic example of the aldo-keto reductase superfamily. The details of the binding of the NADPH cofactor help to explain why 2,5-DKGR exhibits lower binding affinity for cofactor than the related human aldose reductase does. Furthermore, changes in the local loop structure near the cofactor suggest that 2,5-DKGR will not exhibit the biphasic cofactor binding characteristics observed in aldose reductase. Although the crystal structure does not include substrate, the two ordered water molecules present within the substrate-binding pocket are postulated to provide positional landmarks for the substrate 5-keto and 4-hydroxyl groups. The structural basis for several previously described active-site mutants of 2,5-DKGR A is also proposed. Recent research efforts have described a novel approach to the synthesis of L-ascorbate (vitamin C) by using a genetically engineered microorganism that is capable of synthesizing 2,5-DKG from glucose and subsequently is transformed with the gene for 2,5-DKGR. These modifications create a microorganism capable of direct production of 2-keto-L-gulonate from D-glucose, and the gulonate can subsequently be converted into vitamin C. In economic terms, vitamin C is the single most important specialty chemical manufactured in the world. Understanding the structural determinants of specificity, catalysis, and stability for 2,5-DKGR A is of substantial commercial interest. << Less

  Proc. Natl. Acad. Sci. U.S.A. 95:6768-6773(1998) [PubMed] [EuropePMC]