Biochemical and molecular characterization of the isocitrate dehydrogenase with dual coenzyme specificity from the obligate methylotroph Methylobacillus Flagellatus

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.

The Natural History of Cervical Intraepithelial Neoplasia Grades 1, 2, and 3: A Systematic Review and Meta-analysis

“…This resulted in 125 studies being excluded. The remaining 89 studies were included for meta-analysis, of which 63 studies with CIN 1 patients, 8–70 42 studies with CIN 2 patients, 11,26,27,35–37,39,41–44,50,52,53,57,69–95 and 7 studies with CIN 3 patients 3,26,41,72,81,83,88 . The characteristics and the follow-up protocol of the included studies are summarized in Supplementary Tables 1–3, http://links.lww.com/LGT/A239, http://links.lww.com/LGT/A240, http://links.lww.com/LGT/A241.…”

“…High risk of bias was in most studies caused by the unrepresentativeness of the cohort for the full population in a predefined period [11][12][13]24,27,28,30,[32][33][34]36,37,40,[42][43][44]48,50,51,53,57,59,61,71,[73][74][75][76][77]79,[83][84][85][86][87][88][91][92][93]95 and the low number of participants in the study. 8,9,11,12,26,27,34,36,37,39,[42]…”

Synergistic Removal of Nutrient Pollutants and Pharmaceutical and Personal Care Products (PPCPs) from Contaminated Groundwater: Macro- and Microelements and Microorganisms

“…In this study, low concentrations of Fe 3+ (0.1 ± 0.1 mg/L in system E) enhanced the microbial metabolic activity and promoted the degradation of pollutants. Mn 2+ was most effective in enhancing the enzyme activity of Methylophilaceae than Mg 2+ and Zn 2+ . What’s more, Cu 2+ played a key role in the physiology and activity of MOB because it acts as the metal center for the particulate methane monooxygenase of MOB …”

“…Mn 2+ was most effective in enhancing the enzyme activity of Methylophilaceae than Mg 2+ and Zn 2+ . 38 What's more, Cu 2+ played a key role in the physiology and activity of MOB because it acts as the metal center for the particulate methane monooxygenase of MOB. 39 Synergistic removal of nutrient pollutants and PPCPs from contaminated groundwater was achieved in system E; the removal efficiencies of nutrient pollutants were 75.8 ± 20.1%− 94.3 ± 8.8%, which were approximately 29.5 ± 9.8%−39.4 ± 5.4% higher than for the other four systems.…”

Inhibition of hepatic energy metabolizing enzymes in murine model exposed to diisononyl phthalate

“…Romkina and Kiriukhin (2017) previously reported that the decrease in NAD + at 340nm was used to evaluate isocitrate dehydrogenase (IDH) activity. 15…”