Biochemical and molecular characterization of NAD ~+-dependent isocitrate dehydrogenase from the ethanologenic bacterium Zymomonas mobilis

摘要

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.