Enzymatic Characterization of the Isocitrate Dehydrogenase with Dual Coenzyme Specificity from the Marine Bacterium .

Isocitrate dehydrogenase (IDH) can be divided into NAD-dependent and NADP-dependent types based on the coenzyme specificity. It is worth noting that some IDHs exhibit dual coenzyme specificity characteristics. Herein, a dual coenzyme-dependent IDH from (UmIDH) was expressed, purified, and identified in detail for the first time. SDS-PAGE and Gel filtration chromatography analyses showed that UmIDH is an 84.7 kDa homodimer in solution. The values for NAD and NADP are 1800.0 ± 64.4 μM and 1167.7 ± 113.0 μM in the presence of Mn, respectively. Meanwhile, the catalytic efficiency (/) of UmIDH is only 2.3-fold greater for NADP than NAD. The maximal activity for UmIDH occurred at pH 8.5 (with Mn) or pH 8.7 (with Mg) and at 35 °C (with Mn or Mg). Heat inactivation assay revealed that UmIDH sustained 50% of maximal activity after incubation at 57 °C for 20 min with either Mn or Mg. Moreover, three putative core coenzyme binding residues (R345, L346, and V352) of UmIDH were evaluated by site-directed mutagenesis. This recent work identified a unique dual coenzyme-dependent IDH and achieved the groundbreaking bidirectional modification of this specific IDH's coenzyme dependence for the first time. This provides not only a reference for the study of dual coenzyme-dependent IDH, but also a basis for the investigation of the coenzyme-specific evolutionary mechanisms of IDH.