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Biochemical and molecular characterization of the isocitrate dehydrogenase with dual coenzyme specificity from the obligate methylotroph Methylobacillus Flagellatus.
PLoS One. 2017; 12(4):e0176056.Plos

Abstract

The isocitrate dehydrogenase (MfIDH) with unique double coenzyme specificity from Methylobacillus flagellatus was purified and characterized, and its gene was cloned and overexpressed in E. coli as a fused protein. This enzyme is homodimeric,-with a subunit molecular mass of 45 kDa and a specific activity of 182 U mg -1 with NAD+ and 63 U mg -1 with NADP+. The MfIDH activity was dependent on divalent cations and Mn2+ enhanced the activity the most effectively. MfIDH exhibited a cofactor-dependent pH-activity profile. The optimum pH values were 8.5 (NAD+) and 6.0 (NADP+).The Km values for NAD+ and NADP+ were 113 μM and 184 μM respectively, while the Km values for DL-isocitrate were 9.0 μM (NAD+), 8.0 μM (NADP+). The MfIDH specificity (kcat/Km) was only 5-times higher for NAD+ than for NADP+. The purified MfIDH displayed maximal activity at 60°C. Heat-inactivation studies showed that the MfIDH was remarkably thermostable, retaining full activity at 50°C and losting ca. 50% of its activity after one hour of incubation at 75°C. The enzyme was insensitive to the presence of intermediate metabolites, with the exception of 2 mM ATP, which caused 50% inhibition of NADP+-linked activity. The indispensability of the N6 amino group of NAD(P)+ in its binding to MfIDH was demonstrated. MfIDH showed high sequence similarity with bacterial NAD(P)+-dependent type I isocitrate dehydrogenases (IDHs) rather than with eukaryotic NAD+-dependent IDHs. The unique double coenzyme specificity of MfIDH potentially resulted from the Lys340, Ile341 and Ala347 residues in the coenzyme-binding site of the enzyme. The discovery of a type I IDH with double coenzyme specificity elucidates the evolution of this subfamily IDHs and may provide fundamental information for engineering enzymes with desired properties.

Authors+Show Affiliations

Ajinomoto-Genetika Research Institute, Moscow, Russia.Ajinomoto-Genetika Research Institute, Moscow, Russia.

Pub Type(s)

Journal Article

Language

eng

PubMed ID

28423051

Citation

Romkina, Anastasia Y., and Michael Y. Kiriukhin. "Biochemical and Molecular Characterization of the Isocitrate Dehydrogenase With Dual Coenzyme Specificity From the Obligate Methylotroph Methylobacillus Flagellatus." PloS One, vol. 12, no. 4, 2017, pp. e0176056.
Romkina AY, Kiriukhin MY. Biochemical and molecular characterization of the isocitrate dehydrogenase with dual coenzyme specificity from the obligate methylotroph Methylobacillus Flagellatus. PLoS ONE. 2017;12(4):e0176056.
Romkina, A. Y., & Kiriukhin, M. Y. (2017). Biochemical and molecular characterization of the isocitrate dehydrogenase with dual coenzyme specificity from the obligate methylotroph Methylobacillus Flagellatus. PloS One, 12(4), e0176056. https://doi.org/10.1371/journal.pone.0176056
Romkina AY, Kiriukhin MY. Biochemical and Molecular Characterization of the Isocitrate Dehydrogenase With Dual Coenzyme Specificity From the Obligate Methylotroph Methylobacillus Flagellatus. PLoS ONE. 2017;12(4):e0176056. PubMed PMID: 28423051.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Biochemical and molecular characterization of the isocitrate dehydrogenase with dual coenzyme specificity from the obligate methylotroph Methylobacillus Flagellatus. AU - Romkina,Anastasia Y, AU - Kiriukhin,Michael Y, Y1 - 2017/04/19/ PY - 2017/01/23/received PY - 2017/04/04/accepted PY - 2017/4/20/entrez PY - 2017/4/20/pubmed PY - 2017/4/26/medline SP - e0176056 EP - e0176056 JF - PloS one JO - PLoS ONE VL - 12 IS - 4 N2 - The isocitrate dehydrogenase (MfIDH) with unique double coenzyme specificity from Methylobacillus flagellatus was purified and characterized, and its gene was cloned and overexpressed in E. coli as a fused protein. This enzyme is homodimeric,-with a subunit molecular mass of 45 kDa and a specific activity of 182 U mg -1 with NAD+ and 63 U mg -1 with NADP+. The MfIDH activity was dependent on divalent cations and Mn2+ enhanced the activity the most effectively. MfIDH exhibited a cofactor-dependent pH-activity profile. The optimum pH values were 8.5 (NAD+) and 6.0 (NADP+).The Km values for NAD+ and NADP+ were 113 μM and 184 μM respectively, while the Km values for DL-isocitrate were 9.0 μM (NAD+), 8.0 μM (NADP+). The MfIDH specificity (kcat/Km) was only 5-times higher for NAD+ than for NADP+. The purified MfIDH displayed maximal activity at 60°C. Heat-inactivation studies showed that the MfIDH was remarkably thermostable, retaining full activity at 50°C and losting ca. 50% of its activity after one hour of incubation at 75°C. The enzyme was insensitive to the presence of intermediate metabolites, with the exception of 2 mM ATP, which caused 50% inhibition of NADP+-linked activity. The indispensability of the N6 amino group of NAD(P)+ in its binding to MfIDH was demonstrated. MfIDH showed high sequence similarity with bacterial NAD(P)+-dependent type I isocitrate dehydrogenases (IDHs) rather than with eukaryotic NAD+-dependent IDHs. The unique double coenzyme specificity of MfIDH potentially resulted from the Lys340, Ile341 and Ala347 residues in the coenzyme-binding site of the enzyme. The discovery of a type I IDH with double coenzyme specificity elucidates the evolution of this subfamily IDHs and may provide fundamental information for engineering enzymes with desired properties. SN - 1932-6203 UR - https://www.unboundmedicine.com/medline/citation/28423051/Biochemical_and_molecular_characterization_of_the_isocitrate_dehydrogenase_with_dual_coenzyme_specificity_from_the_obligate_methylotroph_Methylobacillus_Flagellatus_ L2 - http://dx.plos.org/10.1371/journal.pone.0176056 DB - PRIME DP - Unbound Medicine ER -