Dataset Information

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

ABSTRACT: 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 Mn²⁺, 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 Mn²⁺) or pH 8.7 (with Mg²⁺) and at 35 °C (with Mn²⁺ or Mg²⁺). Heat inactivation assay revealed that UmIDH sustained 50% of maximal activity after incubation at 57 °C for 20 min with either Mn²⁺ or Mg²⁺. 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.

SUBMITTER: Bian M

PROVIDER: S-EPMC10380307 | biostudies-literature | 2023 Jul

REPOSITORIES: biostudies-literature

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

Bian Mingjie M Zhao Jiaxin J Xu Wenqiang W Han Xueyang X Chen Xuefei X Wang Peng P Zhu Guoping G

International journal of molecular sciences 20230713 14

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 ...[more]

PMID: 37511187

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Project description:Isocitrate dehydrogenase (IDH) family of proteins is classified into three subfamilies, namely, types I, II, and III. Although IDHs are widely distributed in bacteria, archaea, and eukaryotes, all type III IDHs reported to date are found only in prokaryotes. Herein, a novel type III IDH subfamily member from the marine microalga Phaeodactylum tricornutum (PtIDH2) was overexpressed, purified, and characterized in detail for the first time. Relatively few eukaryotic genomes encode this type of IDH and PtIDH2 shares the highest homology with marine bacterial monomeric IDHs, suggesting that PtIDH2 originated through a horizontal gene transfer event between a marine alga and a bacterium. Size-exclusion chromatography revealed that the native PtIDH2 is a homotetramer (∼320 kDa) in solution, comprising four monomeric IDH-like subunits (80 kDa each). Enzymatic characterization showed that PtIDH2 is a bivalent metal ion-dependent enzyme and Mn2+ is the optimal activator. The recombinant PtIDH2 protein exhibited maximal activity at 35°C and pH 8.0 in the presence of Mn2+. Heat-inactivation analysis revealed that PtIDH2 is a cold-adapted enzyme. Kinetic analysis demonstrated that PtIDH2 is a completely NADP+-specific IDH with no detectable NAD+-associated catalytic activity. The three putative key NADP+-binding residues (His604, Arg615, and Arg664) in PtIDH2 were also evaluated by site-directed mutagenesis. The H604L/R615D/R664S triple mutant showed a 3.25-fold preference for NAD+ over NADP+, implying that the coenzyme specificity of PtIDH2 can be converted from NADP+ to NAD+ through rational engineering approaches. Additionally, the roles of the conserved residues Ala718 and Leu742 in the thermostability of PtIDH2 were also explored by site-directed mutagenesis. We found that the L742F mutant displayed higher thermostability than wild-type PtIDH2. This study expands the phylogeny of the IDH family and provides new insights into the evolution of IDHs.

Dataset Information

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

Publications

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

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