Gluconate:NADP 5-oxidoreductase (GNO) from the acetic acid bacterium Gluconobacter oxydans subsp. oxydans DSM3503 was purified to homogeneity. This enzyme is involved in the nonphosphorylative, ketogenic oxidation of glucose and oxidizes gluconate to 5-ketogluconate. GNO was localized in the cytoplasm, had an isoelectric point of 4.3, and showed an apparent molecular weight of 75,000. In sodium dodecyl sulfate gel electrophoresis, a single band appeared corresponding to a molecular weight of 33,000, which indicated that the enzyme was composed of two identical subunits. The pH optimum of gluconate oxidation was pH 10, and apparent K m values were 20.6 mM for the substrate gluconate and 73 M for the cosubstrate NADP. The enzyme was almost inactive with NAD as a cofactor and was very specific for the substrates gluconate and 5-ketogluconate. D-Glucose, D-sorbitol, and D-mannitol were not oxidized, and 2-ketogluconate and L-sorbose were not reduced. Only D-fructose was accepted, with a rate that was 10% of the rate of 5-ketogluconate reduction. The gno gene encoding GNO was identified by hybridization with a gene probe complementary to the DNA sequence encoding the first 20 N-terminal amino acids of the enzyme. The gno gene was cloned on a 3.4-kb DNA fragment and expressed in Escherichia coli. Sequencing of the gene revealed an open reading frame of 771 bp, encoding a protein of 257 amino acids with a predicted relative molecular mass of 27.3 kDa. Plasmidencoded gno was functionally expressed, with 6.04 U/mg of cell-free protein in E. coli and with 6.80 U/mg of cell-free protein in G. oxydans, which corresponded to 85-fold overexpression of the G. oxydans wild-type GNO activity. Multiple sequence alignments showed that GNO was affiliated with the group II alcohol dehydrogenases, or short-chain dehydrogenases, which display a typical pattern of six strictly conserved amino acid residues.Gluconobacter oxydans, a gram-negative, strictly aerobic, rod-shaped bacterium, belongs to the family of acetic acid bacteria (11) and oxidizes glucose via two alternative pathways (28): one requires an initial phosphorylation followed by oxidation via the hexose monophosphate pathway (28); the second involves the nonphosphorylative formation of gluconic acid and of ketogluconic acids (12,22). The enzyme catalyzing the formation of gluconic acid, glucose dehydrogenase, is located in the cytoplasmic membrane of G. oxydans and is coupled to the respiratory chain (3). Further oxidation of gluconic acid by the membrane-bound and respiratory chain-coupled enzymes gluconate dehydrogenase and 2-ketogluconate dehydrogenase leads to the formation of 2-ketogluconic and 2,5-diketogluconic acids (39, 40). The enzymatic activity converting gluconic acid to 5-ketogluconic acid, however, is located in the cytoplasm of G. oxydans and depends on the presence of the cofactor NADP (12, 27). The enzymes catalyzing this reaction have been purified from G. suboxydans and Gluconobacter liquefaciens (1, 2). However, these enzymes were termed 5-ketogluconate...

The pyruvate dehydrogenase (PDH) complex of the gram-negative bacterium Zymomonas mobilis was purified to homogeneity. From 250 g of cells, we isolated 1 mg of PDH complex with a specific activity of 12.6 U/mg of protein. Analysis of subunit composition revealed a PDH (E1) consisting of the two subunits E1α (38 kDa) and E1β (56 kDa), a dihydrolipoamide acetyltransferase (E2) of 48 kDa, and a lipoamide dehydrogenase (E3) of 50 kDa. The E2 core of the complex is arranged to form a pentagonal dodecahedron, as shown by electron microscopic images, resembling the quaternary structures of PDH complexes from gram-positive bacteria and eukaryotes. The PDH complex-encoding genes were identified by hybridization experiments and sequence analysis in two separate gene regions in the genome ofZ. mobilis. The genes pdhAα (1,065 bp) andpdhAβ (1,389 bp), encoding the E1α and E1β subunits of the E1 component, were located downstream of the gene encoding enolase. The pdhB (1,323 bp) and lpd (1,401 bp) genes, encoding the E2 and E3 components, were identified in an unrelated gene region together with a 450-bp open reading frame (ORF) of unknown function in the order pdhB-ORF2-lpd. Highest similarities of the gene products of the pdhAα,pdhAβ, and pdhB genes were found with the corresponding enzymes of Saccharomyces cerevisiae and other eukaryotes. Like the dihydrolipoamide acetyltransferases of S. cerevisiae and numerous other organisms, the product of thepdhB gene contains a single lipoyl domain. The E1β subunit PDH was found to contain an amino-terminal lipoyl domain, a property which is unique among PDHs.

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The dependence of tartaric acid production by Gluconobacter oxydans ssp. oxydans ATCC 19357 and G. oxydans ssp. suboxydans ATCC 621 on vanadate was investigated. It was found with both organisms that trataric acid could only be produced in a medium containing vanadate (NH4VO3). A proposed intermediate of the tartaric acid metabolism in G. oxydans, 5‐ketogluconic acid, was tested on its reactivity in the presence of the oxidizing catalyst vanadate. It could be shown that 5‐ketogluconic acid and the catalyst vanadate, but not the activity of G. oxydans, were responsible for the formation of tartaric acid. G. oxydans was not able to produce tartaric acid by itself. The stereochemical identity of the formed tartaric acid could be identified as the L‐(+)‐type. Oxalic acid was formed from 5‐ketogluconic acid with vanadate in the absence and in the presence of G. oxydans. The ratio of oxalic acid to tartaric acid was 1:1.