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

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 Umbonibacter Marinipuiceus (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 K _m values for NAD ⁺ and NADP ⁺ are 1800.0 ± 64.4 μM and 1167.7 ± 113.0 μM in the presence of Mn ²⁺ , respectively. Meanwhile, the catalytic efficiency ( k _cat / K _m ) of UmIDH is only 2.3-fold greater for NADP ⁺ than NAD ⁺ . The maximal activity for UmIDH occurred at pH 8.5 (with Mn ²⁺ ) or pH 8.7 (with Mg ²⁺ ) and at 35 °C (with Mn ²⁺ or Mg ²⁺ ). Heat inactivation assay revealed that UmIDH sustained 50% of maximal activity after incubation at 57 °C for 20 min with either Mn ²⁺ or Mg ²⁺ . 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.