title: Biochemical and molecular characterization of NAD(+)-dependent isocitrate dehydrogenase from the ethanologenic bacterium Zymomonas mobilis.
abstract: An isocitrate dehydrogenase from Zymomonas mobilis was overexpressed in Escherichia coli as a fused protein (ZmIDH). The molecular mass of recombinant ZmIDH, together with its 6× His partner, was estimated to be 74 kDa by gel filtration chromatography, suggesting a homodimeric structure. The purified recombinant ZmIDH displayed maximal activity at 55 °C, pH 8.0 with Mn(2+) and pH 8.5 with Mg(2+). Heat inactivation studies showed that the recombinant ZmIDH was rapidly inactivated above 40 °C. In addition, the recombinant ZmIDH activity was completely dependent on the divalent cation and Mn(2+) was the most effective cation. The recombinant ZmIDH displayed a 165-fold (k(cat)/K(m)) preference for NAD(+) over NADP(+) with Mg(2+), and a 142-fold greater specificity for NAD(+) than NADP(+) with Mn(2+). Therefore, the recombinant ZmIDH has remarkably high coenzyme preference for NAD(+). The catalytic efficiency (k(cat)/K(m)) of the recombinant ZmIDH was found to be much lower than that of its NADP(+)-dependent counterparts. The poor performance of the recombinant ZmIDH in decarboxylating might be improved by protein engineering techniques, thus making ZmIDH a potential genetic modification target for the development of optimized Z. mobilis strains.
authors: The Key Laboratory of Molecular Evolution and Biodiversity, Institute of Molecular Biology and Biotechnology, College of Life Sciences, Anhui Normal University, Wuhu, China.
authors: Peng
authors: P
authors: Wang
authors: Mingming
authors: M
authors: Jin
authors: Guoping
authors: G
authors: Zhu
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability; D007521:Isocitrate Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D013379:Substrate Specificity; D017355:Zymomonas / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D007521:Isocitrate Dehydrogenase