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. © 2011 Federation of European Microbiological Societies.