Biochemical characterization and sequence analysis of the gluconate:NADP 5-oxidoreductase gene from Gluconobacter oxydans

Abstract

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...

Structural insight into the catalytic mechanism of gluconate 5‐dehydrogenase from Streptococcus suis: Crystal structures of the substrate‐free and quaternary complex enzymes

“…These results provide evidence that the Ga5DH from S. suis functions as a homotetramer, in contrast with the dimeric homologous proteins from Thermotoga maritima and Gluconobacter oxydans. 5 The tetrameric form of Ga5DH in the solution-state is consistent with that deduced from the solid-state crystal structure.…”

“…4 Under conditions of energy surplus, the enzyme catalyzes the oxidation of D-gluconate to 5-keto-D-gluconate as a transient means of energy storage. 5 The reactions catalyzed by Ga5DH have been confirmed by HPLC and NMR analysis. 6 The NADPH generated during the oxidation of D-gluconate may function as a hydrogen donor for biosynthetic processes, and it may also play a pivotal role in the defense of the organism against the oxidative attack by the infected host.…”

“…6 The NADPH generated during the oxidation of D-gluconate may function as a hydrogen donor for biosynthetic processes, and it may also play a pivotal role in the defense of the organism against the oxidative attack by the infected host. 7 To date, biochemical characteristics have been reported for Ga5DHs from several species including, Gluconobacter oxydans, 5,8 Gluconobacter liquefaciens, 9 and Acetobacter suboxydans. 10 The enzyme belongs to the short chain dehydrogenase/reductase (SDR) family with a preference for NADP þ rather than NAD þ as cofactor.…”

“…10 The enzyme belongs to the short chain dehydrogenase/reductase (SDR) family with a preference for NADP þ rather than NAD þ as cofactor. 5 The crystal structure of Ga5DH in complex with NADP þ from Thermotoga maritima (PDB code 1VL8) has been solved, but the data have not yet been published. Consequently, no structural information for any Ga5DH is currently available to permit elucidation of the molecular architecture or clarification of the catalytic mechanism of the enzyme.…”

Acetic Acid Bacteria: Physiology and Carbon Sources Oxidation

“…This reaction is particularly active in Gluconobacter growing at high concentrations of sugars. D-gluconate can be further oxidized to 2-ketogluconate and 2,5-diketogluconate by the gluconate dehydrogenase and 2-ketogluconate dehydrogenase [52].…”