An isocitrate dehydrogenase (ICDH) with an unique coenzyme specificity from Acidithiobacillus thiooxidans was purified and characterized, and its gene was cloned. The native enzyme was homodimeric with a subunit of M(r) 45000 and showed a 78-fold preference for NAD(+) over NADP(+). The cloned ICDH gene (icd) was expressed in an icd-deficient strain of Escherichia coli EB106; the activity was found in the cell extract. The gene encodes a 429-amino acid polypeptide and is located between open reading frames encoding a putative aconitase gene (upstream of icd) and a putative succinyl-CoA synthase beta-subunit gene (downstream of icd). A. thiooxidans ICDH showed high sequence similarity to bacterial NADP(+)-dependent ICDH rather than eukaryotic NAD(+)-dependent ICDH, but the NAD(+)-preference of the enzyme was suggested due to residues conserved in the coenzyme binding site of the NAD(+)-dependent decarboxylating dehydrogenase.
SummaryVolatile sulfur compounds (VS) are generated in the large intestine by the bac terial metabolism of sulfate and sulfur amino acids. VS are potentially harmful to the host. The effect of dietary supplementation of herb extracts on volatile sulfur production in the large intestine of pig was evaluated in this study. The extracts Perilla frutescens (Soyou), Mentha piperita (Peppermint), and Ajuga decumbens (Kiransou) were fed to pigs equipped with a permanent cannula at the cecum. Cecal digesta were sampled and analyzed for am monia and short-chain fatty acids (SCFA). Sampled digesta were incubated anaerobically either with or without L-methionine for 24h to estimate volatile sulfur production in vivo. L-Methionine was supplemented to enhance methanethiol (MeSH) production. At the end of the incubation, head space concentrations of volatile sulfur compounds such as hydrogen sulfide (H2S), MeSH, and dimethyl sulfide (DMS) were determined by flame-photometric gaschromatography after the addition of 6N HCl. Sampled digesta were also subjected to the most probable number estimations for sulfate-reducing bacteria (SRB), sulfide producer from L-methionine, and MeSH producers from L-methionine. All three herb extracts signifi cantly decreased H2S (p<0.05), MeSH (p<0.05), and ammonia (p<0.05) production, but SCFA production was not affected (p>0.05). The number of volatile sulfur-producing bacte ria did not vary among groups by the dietary supplementation of these herb extracts. Serial solvent extraction was done on these herb extracts to specify the active fractions that reduce volatile sulfur production, n-Butanol fraction of all three extracts significantly reduced volatile sulfur production in vitro.
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