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DNA from uncultured organisms as a source of 2,5-diketo-D-gluconic acid reductases.

ABSTRACT: Total DNA of a population of uncultured organisms was extracted from soil samples, and by using PCR methods, the genes encoding two different 2,5-diketo-D-gluconic acid reductases (DKGRs) were recovered. Degenerate PCR primers based on published sequence information gave internal gene fragments homologous to known DKGRs. Nested primers specific for the internal fragments were combined with random primers to amplify flanking gene fragments from the environmental DNA, and two hypothetical full-length genes were predicted from the combined sequences. Based on these predictions, specific primers were used to amplify the two complete genes in single PCRs. These genes were cloned and expressed in Escherichia coli. The purified gene products catalyzed the reduction of 2,5-diketo-D-gluconic acid to 2-keto-L-gulonic acid. Compared to previously described DKGRs isolated from Corynebacterium spp., these environmental reductases possessed some valuable properties. Both exhibited greater than 20-fold-higher kcat/Km values than those previously determined, primarily as a result of better binding of substrate. The Km values for the two new reductases were 57 and 67 microM, versus 2 and 13 mM for the Corynebacterium enzymes. Both environmental DKGRs accepted NADH as well as NADPH as a cosubstrate; other DKGRs and most related aldo-keto reductases use only NADPH. In addition, one of the new reductases was more thermostable than known DKGRs.

SUBMITTER: Eschenfeldt WH

PROVIDER: S-EPMC93149 | biostudies-literature | 2001 Sep

REPOSITORIES: biostudies-literature

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DNA from uncultured organisms as a source of 2,5-diketo-D-gluconic acid reductases.

Eschenfeldt W H WH Stols L L Rosenbaum H H Khambatta Z S ZS Quaite-Randall E E Wu S S Kilgore D C DC Trent J D JD Donnelly M I MI

Applied and environmental microbiology 20010901 9

Total DNA of a population of uncultured organisms was extracted from soil samples, and by using PCR methods, the genes encoding two different 2,5-diketo-D-gluconic acid reductases (DKGRs) were recovered. Degenerate PCR primers based on published sequence information gave internal gene fragments homologous to known DKGRs. Nested primers specific for the internal fragments were combined with random primers to amplify flanking gene fragments from the environmental DNA, and two hypothetical full-len ...[more]

PMID: 11526025

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Project description: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.

Project description:BackgroundCurrent understanding of the carbon cycle in methanogenic environments involves trophic interactions such as interspecies H2 transfer between organotrophs and methanogens. However, many metabolic processes are thermodynamically sensitive to H2 accumulation and can be inhibited by H2 produced from co-occurring metabolisms. Strategies for driving thermodynamically competing metabolisms in methanogenic environments remain unexplored.ResultsTo uncover how anaerobes combat this H2 conflict in situ, we employ metagenomics and metatranscriptomics to revisit a model ecosystem that has inspired many foundational discoveries in anaerobic ecology-methanogenic bioreactors. Through analysis of 17 anaerobic digesters, we recovered 1343 high-quality metagenome-assembled genomes and corresponding gene expression profiles for uncultured lineages spanning 66 phyla and reconstructed their metabolic capacities. We discovered that diverse uncultured populations can drive H2-sensitive metabolisms through (i) metabolic coupling with concurrent H2-tolerant catabolism, (ii) forgoing H2 generation in favor of interspecies transfer of formate and electrons (cytochrome- and pili-mediated) to avoid thermodynamic conflict, and (iii) integration of low-concentration O2 metabolism as an ancillary thermodynamics-enhancing electron sink. Archaeal populations support these processes through unique methanogenic metabolisms-highly favorable H2 oxidation driven by methyl-reducing methanogenesis and tripartite uptake of formate, electrons, and acetate.ConclusionIntegration of omics and eco-thermodynamics revealed overlooked behavior and interactions of uncultured organisms, including coupling favorable and unfavorable metabolisms, shifting from H2 to formate transfer, respiring low-concentration O2, performing direct interspecies electron transfer, and interacting with high H2-affinity methanogenesis. These findings shed light on how microorganisms overcome a critical obstacle in methanogenic carbon cycles we had hitherto disregarded and provide foundational insight into anaerobic microbial ecology. Video Abstract.

Dataset Information

DNA from uncultured organisms as a source of 2,5-diketo-D-gluconic acid reductases.

Publications

DNA from uncultured organisms as a source of 2,5-diketo-D-gluconic acid reductases.

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