The structure of allophanate hydrolase from Granulibacter bethesdensis provides insights into substrate specificity in the amidase signature family.
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Cloning and nucleotide sequencing of the membrane-bound L-sorbosone dehydrogenase gene of Acetobacter liquefaciens IFO 12258 and its expression in Gluconobacter oxydans.
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Cloning and expression of the gene encoding Acetobacter liquefaciens IFO 12258 membrane-bound L-sorbosone dehydrogenase (SNDH) were studied. A genomic library of A. liquefaciens IFO 12258 was constructed with the mobilizable cosmid vector pVK102 (mob+) in Escherichia coli S17-1 (Tra+). The library was transferred by conjugal mating into Gluconobacter oxydans OX4, a mutant of G. oxydans IFO 3293 that accumulates L-sorbosone in the presence of L-sorbose. The transconjugants were screened for SNDH activity by performing a direct expression assay. One clone harboring plasmid p7A6 converted L-sorbosone to 2-keto-L-gulonic acid (2KGA) more rapidly than its host did and also converted L-sorbose to 2KGA with no accumulation of L-sorbosone. The insert (25 kb) of p7A6 was shortened to a 3.1-kb fragment, in which one open reading frame (1,347 bp) was found and was shown to encode a polypeptide with a molecular weight of 48,222. The SNDH gene was introduced into the 2KGA-producing strain G. oxydans IFO 3293 and its derivatives, which contained membrane-bound L-sorbose dehydrogenase. The cloned SNDH was correctly located in the membrane of the host. The membrane fraction of the clone exhibited almost stoichiometric formation of 2KGA from L-sorbosone and L-sorbose. Resting cells of the clones produced 2KGA very efficiently from L-sorbosone and L-sorbose, but not from D-sorbitol; the conversion yield from L-sorbosone was improved from approximately 25 to 83%, whereas the yield from L-sorbose was increased from 68 to 81%. Under fermentation conditions, cloning did not obviously improve the yield of 2KGA from L-sorbose. (+info)
Generation mechanism and purification of an inactive form convertible in vivo to the active form of quinoprotein alcohol dehydrogenase in Gluconobacter suboxydans.
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Alcohol dehydrogenase (ADH) of acetic acid bacteria is a membrane-bound quinohemoprotein-cytochrome c complex involved in vinegar production. In Gluconobacter suboxydans grown under acidic growth conditions, it was found that ADH content in the membranes was largely increased but the activity was not much changed, suggesting that such a condition produces an inactive form of ADH (inactive ADH). A similar phenomenon could be also observed in Acetobacter aceti, another genus of acetic acid bacteria. Furthermore, aeration conditions were also shown to affect ADH production; the ADH level was increased and was present as an active form under low-aeration conditions, while the ADH level was decreased and was present mainly as an inactive form under high-aeration conditions. Inactive ADH was solubilized from the membranes of G. suboxydans grown in acidic and high-aeration conditions and was purified separately from the normal, active form of ADH (active ADH). In spite of having 10 times less enzyme activity than active ADH, inactive ADH could not be distinguished from active ADH with respect to their subunit compositions, molecular sizes, and prosthetic groups. Inactive ADH, however, had a relatively loose conformation with a partially oxidized state, while active ADH had a tight conformation with a completely reduced state, suggesting that inactive ADH may lack a right subunit's interaction and that one of the heme c components may be inactivated. Reactivation from such an inactive ADH occurred either by shifting of the pH of the culture medium up during the cultivation or by incubation of the resting cells at the neutral pH region in the presence of an energy source such as D-sorbitol. Such an activation of ADH was repressed by the addition of a proton uncoupler and could not occur in the spheroplasts. Thus, the results suggest that inactive ADH could be generated abundantly under acidic growth conditions and converted to the active form at a neutral culture pH. The data also suggest that some periplasmic component may be involved in the conversion of inactive ADH into the active form by consuming some forms of energy. (+info)