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.

Fig 1. Overexpression, purification and oligomeric state determination of the recombinant MfIDH.

(a) The protein purity was determined using 12% SDS-PAGE. M, protein marker; lane 1, crude extracts of cells harboring plasmid pET-MfIDH after induction with IPTG; lane 2, purified protein. (b) Detection of MfIDH by Western blot using the anti-6×His antibody as a probe. Lane 1, negative control, crude extracts of cells harboring pET-15b(+) with IPTG induction; lane 2, purified protein. (c) Gradient non-denaturing PAGE. M, protein marker; lane 1, purified native MfIDH; lane 2, purified recombinant MfIDH; Zymogram assay of the purified proteins. Staining for the NADP⁺-dependent activity: lane 3, native MfIDH, lane 4, recombinant MfIDH. Staining for the NAD⁺-dependent activity: lane 5, native MfIDH, lane 6, recombinant MfIDH. (d) Molecular mass determination using gel filtration chromatography. The flow rate was 0.5 mL min^-1, and the proteins were detected by monitoring their absorbance at 280 nm. The molecular mass standard curve is inset. The measurement of the recombinant MfIDH is represented as a dark dot (●). The standard proteins are represented as open circles (○) and are carbonic anhydrase (29 kDa), albumin (66 kDa), alcohol dehydrogenase (150 kDa), β-amylase (200 kDa), apoferritin (443 kDa) and thyroglobulin (669 kDa). The V_e of the recombinant MfIDH is 13.36 mL.

Fig 2. Structure-based sequence alignment of MfIDH with other dimeric IDHs.

High-resolution crystal structures of the A. thiooxidans NAD-IDH (AtIDH, 2D4V), B. subtilis NADP-IDH (BsIDH, 1HQS) and E. coli NADP-IDH (EcIDH, 9ICD) were downloaded from the PDB database. The MfIDH model of the IDH from M. flagellatus and the ZmIDH model of the NAD-IDH from Z. mobilis were generated using the SWISS-MODEL modeling server with AtIDH structure as the template. The secondary structure of MfIDH is depicted above the alignment. The completely conserved amino acids are highlighted as shaded red boxes. The conserved residues involved in cofactor- (●) and substrate-binding (▲) are indicated, respectively. The conserved phosphorylation sites are indicated (■). The lysine residues that may be acetylated in MfIDH and EcIDH are highlighted with shades light-blue and pink boxes, respectively. The major cofactor specificity determinants are highlighted with shaded yellow boxes and indicated with stars (★). The alignment was drawn with ESPRIPT 3.0.

Fig 3. Effects of pH and temperature on the activity of MfIDH.

(a) The effects of pH on the NAD⁺-dependent (●) and NADP⁺-dependent (○) activities of MfIDH from pH 5.0 to 10.0 in the presence of Mn²⁺. (b) The effects of temperature on NAD⁺-dependent (●) and NADP⁺-dependent (○) activities of MfIDH from 45 to 65°C. (c) Heat-inactivation profiles of NAD⁺-dependent (●) and NADP⁺-dependent (○) activities of MfIDH incubated at 50 to 80°C. The incubation time is 60 min.