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Biochemical Characterization of Recombinant Isocitrate Dehydrogenase and Its Putative Role in the Physiology of an Acidophilic Micrarchaeon.

ABSTRACT: Despite several discoveries in recent years, the physiology of acidophilic Micrarchaeota, such as "Candidatus Micrarchaeum harzensis A_DKE", remains largely enigmatic, as they highly express numerous genes encoding hypothetical proteins. Due to a lacking genetic system, it is difficult to elucidate the biological function of the corresponding proteins and heterologous expression is required. In order to prove the viability of this approach, A_DKE's isocitrate dehydrogenase (MhIDH) was recombinantly produced in Escherichia coli and purified to electrophoretic homogeneity for biochemical characterization. MhIDH showed optimal activity around pH 8 and appeared to be specific for NADP⁺ yet promiscuous regarding divalent cations as cofactors. Kinetic studies showed K_M-values of 53.03 ± 5.63 µM and 1.94 ± 0.12 mM and k_cat-values of 38.48 ± 1.62 and 43.99 ± 1.46 s^-1 resulting in k_cat/K_M-values of 725 ± 107.62 and 22.69 ± 2.15 mM^-1 s^-1 for DL-isocitrate and NADP⁺, respectively. MhIDH's exceptionally low affinity for NADP⁺, potentially limiting its reaction rate, can likely be attributed to the presence of a proline residue in the NADP⁺ binding pocket, which might cause a decrease in hydrogen bonding of the cofactor and a distortion of local secondary structure.

SUBMITTER: Winkler D

PROVIDER: S-EPMC8623467 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description: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coli to initiate its expression in the glutamate auxotrophic strain E. coli Δicd::kan(r) of which the icd gene has been replaced by kanamycin resistance gene.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 Mn(2+) 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 Km 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.

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Biochemical Characterization of Recombinant Isocitrate Dehydrogenase and Its Putative Role in the Physiology of an Acidophilic Micrarchaeon.

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