Journal Article

Biochemical and molecular characterization of NAD<sup>+</sup>-dependent isocitrate dehydrogenase from the ethanologenic bacterium <i>Zymomonas mobilis</i>

Peng Wang, Mingming Jin and Guoping Zhu

in FEMS Microbiology Letters

Volume 327, issue 2, pages 134-141
Published in print February 2012 |
Published online January 2012 | e-ISSN: 1574-6968 | DOI:

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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 Mn2+ and pH 8.5 with Mg2+. 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 Mn2+ was the most effective cation. The recombinant ZmIDH displayed a 165-fold (kcat/Km) preference for NAD+ over NADP+ with Mg2+, and a 142-fold greater specificity for NAD+ than NADP+ with Mn2+. Therefore, the recombinant ZmIDH has remarkably high coenzyme preference for NAD+. The catalytic efficiency (kcat/Km) 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.

Keywords: isocitrate dehydrogenase; coenzyme specificity; catalytic efficiency; Zymomonas mobilis

Journal Article.  3864 words.  Illustrated.

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