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.

No abstract

Isocitrate deyhdrogenase (IDH) is a reversible enzyme in the tricarboxylic acid cycle that catalyzes the NAD(P)+-dependent oxidative decarboxylation of isocitrate to α-ketoglutarate (αKG) and the NAD(P)H/CO2-dependent reductive carboxylation of αKG to isocitrate. The IDH gene from Streptococcus mutans was fused with the icd gene promoter from Escherichia coli to initiate its expression in the glutamate auxotrophic strain E. coli Δicd::kanr of which the icd gene has been replaced by kanamycin resistance gene. The expression of S. mutans IDH (SmIDH) may restore the wild-type phenotype of the icd-defective strain on minimal medium without glutamate. The molecular weight of SmIDH was estimated to be 70 kDa by gel filtration chromatography, suggesting a homodimeric structure. SmIDH was divalent cation-dependent and Mn2+ was found to be the most effective cation. The optimal pH of SmIDH was 7.8 and the maximum activity was around 45°C. SmIDH was completely NAD+ dependent and its apparent K m for NAD+ was 137 μM. In order to evaluate the role of the putative phosphorylation site at Ser102 in catalysis, two “stably phosphorylated” mutants were constructed by converting Ser102 into Glu102 or Asp102 in SmIDH to mimick a constitutively phosphorylated state. Meanwhile, the functional roles of another four amino acids (threonine, glycine, alanine and tyrosine) containing variant size of side chains were investigated. The replacement of Asp102 or Glu102 totally inactivated the enzyme, while the S102T, S102G, S102A and S102Y mutants decreased the affinity to isocitrate and only retained 16.0%, 2.8%, 3.3% and 1.1% of the original activity, respectively. These results reveal that Ser102 plays important role in substrate binding and is required for the enzyme function. Also, Ser102 in SmIDH is a potential phosphorylation site, indicating that the ancient NAD-dependent IDHs might be the underlying origin of “phosphorylation mechanism” used by their bacterial NADP-dependent homologs.

Microcystis aeruginosa is the key symptom of water eutrophication and produces persistent microcystins. Our special attention was paid to the isocitrate dehydrogenase (IDH) of M. aeruginosa (MaIDH) because it plays important roles in energy and biosynthesis metabolisms and its catalytic product 2-oxoglutarate provides the carbon skeleton for ammonium assimilation and also constitutes a signaling molecule of nitrogen starvation in cyanobacteria. Sequence alignment showed that MaIDH shared significant sequence identity with IDHs from other cyanobacteria (>80 %) and other bacteria (>45 %). The subunit molecular weight of MaIDH was determined to be 52.6 kDa by filtration chromatography, suggesting MaIDH is a typical homodimer. The purified recombinant MaIDH was completely NADP(+)-dependent and no NAD(+)-linked activity was detectable. The K m values for NADP(+) were 32.24 and 71.71 μM with Mg(2+) and Mn(2+) as a sole divalent cation, and DL-isocitrate linked K m values were 32.56 μM (Mg(2+)) and 124.3 μM (Mn(2+)), respectively. As compared with Mn(2+), MaIDH showed about 2.5-times and 4-times higher affinities (1/K m) to NADP(+) and DL-isocitrate with Mg(2+). The optimum activity of MaIDH was found at pH 7.5, and its optimum temperature was 45 °C (Mn(2+)) and 50 °C (Mg(2+)). Heat-inactivation studies showed that heat treatment for 20 min at 45 °C caused a 50 % loss of enzyme activity. MaIDH was completely divalent cation dependent as other typical dimeric IDHs and Mn(2+) was its best activator. Our study is expected to give a better understanding of primary metabolic enzymes in M. aeruginosa. This would provide useful basic information for the research of controlling the blue-green algae blooms through biological techniques.

Many isocitrate dehydrogenases (IDHs) are dimeric enzymes whose catalytic sites are located at the intersubunit interface, whereas monomeric IDHs form catalytic sites with single polypeptide chains. It was proposed that monomeric IDHs were evolved from dimeric ones by partial gene duplication and fusion, but the evolutionary process had not been reproduced in laboratory. To construct a chimeric monomeric IDH from homo‐dimeric one, it is necessary to reconstitute an active center by a duplicated region; to properly link the duplicated region to the rest part; and to optimize the newly formed protein surface. In this study, a chimeric monomeric IDH was successfully constructed by using homo‐dimeric Escherichia coli IDH as a start point by rational design and site‐saturation mutagenesis. The ~67 kDa chimeric enzyme behaved as a monomer in solution, with a Km of 61 μM and a kcat of 15 s−1 for isocitrate in the presence of NADP+ and Mn2+. Our result demonstrated that dimeric IDHs have a potential to evolve monomeric ones. The evolution of the IDH family was also discussed.