Publications

• A novel oxalosuccinate-forming enzyme involved in the reductive carboxylation of 2-oxoglutarate in Hydrogenobacter thermophilus TK-6.

  Aoshima M., Igarashi Y.

  We have previously demonstrated that the reductive carboxylation of 2-oxoglutarate in Hydrogenobacter thermophilus TK-6 is not simply a reversal of the oxidative decarboxylation catalysed by isocitrate dehydrogenase (ICDH). The reaction involves a novel biotin protein (carboxylating factor for ICD ... >> More

  We have previously demonstrated that the reductive carboxylation of 2-oxoglutarate in Hydrogenobacter thermophilus TK-6 is not simply a reversal of the oxidative decarboxylation catalysed by isocitrate dehydrogenase (ICDH). The reaction involves a novel biotin protein (carboxylating factor for ICDH-CFI) and ATP. In this study, we have analysed the ICDH/CFI system responsible for the carboxylation reaction. Sequence analysis revealed a close relationship between CFI and pyruvate carboxylase. Rather unexpectedly, the rate of ATP hydrolysis was greater than that of isocitrate formation or NADH oxidation. Furthermore, ATP hydrolysis catalysed by CFI was dependent on 2-oxoglutarate but not on ICDH, suggesting that a carboxylated product is formed in the absence of ICDH. The product, which was detectable only at low temperatures, was identified as oxalosuccinate. Thus, CFI was confirmed to be a novel enzyme that catalyses the carboxylation of 2-oxoglutarate to form oxalosuccinate, which corresponds to the first step of the reductive carboxylation from 2-oxoglutarate to isocitrate. The CFI-ICDH system may also be present in mammals, where it could play a significant role in modulating central metabolism. << Less

  Mol. Microbiol. 62:748-759(2006) [PubMed] [EuropePMC]

• A novel biotin protein required for reductive carboxylation of 2-oxoglutarate by isocitrate dehydrogenase in Hydrogenobacter thermophilus TK-6.

  Aoshima M., Ishii M., Igarashi Y.

  Isocitrate dehydrogenase was purified from Hydrogenobacter thermophilus, and the corresponding gene was cloned and sequenced. The enzyme had similar structural properties to the isocitrate dehydrogenase of Escherichia coli, but differed in its catalytic properties, such as coenzyme specificity, pH ... >> More

  Isocitrate dehydrogenase was purified from Hydrogenobacter thermophilus, and the corresponding gene was cloned and sequenced. The enzyme had similar structural properties to the isocitrate dehydrogenase of Escherichia coli, but differed in its catalytic properties, such as coenzyme specificity, pH dependency and kinetic parameters. Notably, the enzyme catalysed the oxidative decarboxylation of isocitrate, but not the reductive carboxylation of 2-oxoglutarate. The carboxylation reaction required the addition of cell extract and ATP-Mg, suggesting the existence of additional carboxylation factor(s). Further analysis of the carboxylation factor(s) resulted in the purification of two polypeptides. N-terminal amino acid sequencing revealed that the two polypeptides are homologues of pyruvate carboxylase with a biotinylated subunit, but do not catalyse pyruvate carboxylation. Pyruvate carboxylase was also purified, but was not active in stimulating isocitrate dehydrogenase. Isocitrate dehydrogenase, the novel biotin protein, ATP-Mg and NADH were essential for the reductive carboxylation of 2-oxoglutarate. These observations indicate that the novel biotin protein is an ATP-dependent factor, which is involved in the reverse (carboxylating) reaction of isocitrate dehydrogenase. << Less

  Mol. Microbiol. 51:791-798(2004) [PubMed] [EuropePMC]