BIOCHEMICAL-CHARACTERIZATION AND SEQUENCE-ANALYSIS OF THE GLUCONATE-NADP 5-OXIDOREDUCTASE GENE FROM GLUCONOBACTER-OXYDANS

Gluconate:NADP 5-oxidoreductase (GNO) from the acetic acid bacterium G luconobacter oxydans subsp. oxydans DSM3503 was purified to homogeneit y. This enzyme is involved in the nonphosphorylative, ketogenic oxidat ion of glucose and oxidizes gluconate to 5-ketogluconate. GNO was loca lized in the cytoplasm, had an isoelectric point of 4.3, and showed an apparent molecular weight of 75,000. In sodium dodecyl sulfate gel el ectrophoresis, a single band appeared corresponding to a molecular wei ght of 33,000, which indicated that the enzyme was composed of two ide ntical 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 mu M for the cosubstrate NADP. The enzyme was almost inactive with NAD a s 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 w ith 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 t he gene revealed an open reading frame of 771 bp, encoding a protein o f 257 amino acids with a predicted relative molecular mass of 27.3 kDa . Plasmid-encoded gno was functionally expressed, with 6.04 U/mg of ce ll-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. oxy dans 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.