Isocitrate Dehydrogenase from Streptococcus mutans: Biochemical Properties and Evaluation of a Putative Phosphorylation Site at Ser102

Abstract

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coli to initiate its expression in the glutamate auxotrophic strain E. coli Δicd::kanr of which the icd gene has been replaced by kanamycin resistance gene.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 Mn2+ 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 K m 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.

Enzymatic characterization and functional implication of two structurally different isocitrate dehydrogenases from Xylella fastidiosa

“…Kinetic studies have shown that the K m value of diXfIDH for NAD + (82 μM) was significantly lower than similar type IDHs, such as those from Methylococcus capsulatus (122 μM) , Hydrogenobacter thermophilus (357 μM), and Streptococcus mutans (154 μM) , but higher than Pyrococcus furiosus (68.3 μM) and the NAD‐using monomeric IDH from Campylobacter sp. FOBRC14 (29 μM) .…”

Phosphoproteomic analysis of bacillus Calmette–Guérin using gel-based and gel-free approaches

“…Here two phosphorylated peptides were identified in DLAT. Isocitrate dehydrogenase could have an important impact on the growth and pathogenesis of the bacteria, such as M. tuberculosis [55] and Streptococcus mutans [56], and this enzyme could be considered possible candidates for use in the serological diagnostics of tuberculosis.…”

A unique homodimeric NAD + ‐linked isocitrate dehydrogenase from the smallest autotrophic eukaryote Ostreococcus tauri

“…Ellipticity (θ) was generated by performing an average of 3 scans per sample in a 0.1 cm path length cell between 195 and 260 nm at 1 nm increments. The calculation of mean residue ellipticity, [θ] (deg cm 2 dmol ‐1 ), has been described elsewhere (11).…”

“…Ellipticity (u) was generated by performing an average of 3 scans per sample in a 0.1 cm path length cell between 195 and 260 nm at 1 nm increments. The calculation of mean residue ellipticity, [u]( d e gc m 2 dmol 21 ), has been described elsewhere (11). A) The N-terminal sequence of OtIDH (XP_003082777) was aligned with mitochondrial NADP-IDHs from S. cerevisiae (ScIDP1, P21954) and porcine heart (PcIDH, P33198), and cytosolic NADP-IDHs from potato (StIDH, P50217) and tobacco (NcIDH, P50218), based on ScIDP1 structure (PDB entry 2QFV).…”

“…Eukaryotes usually possess a NAD + ‐dependent IDH (NAD‐IDH, EC 1.1.1.41) that is strictly mitochondrial and several types of NADP + ‐dependent IDH (NADP‐IDH, EC 1.1.1.42) isoforms that are distributed in the mitochondrial matrix, cell cytosol, and peroxisome (2–5). Mitochondrial NAD‐IDHs are believed to be heteromeric in solution, such as the hetero‐octameric yeast NAD‐IDH, composed of 4 heterodimers of regulatory IDH1 and catalytic IDH2 subunits (6), and the heterotetrameric human NAD‐IDH, with 3 types of subunits present in the ratio of 2α:1β:1γ (7–9), whereas most prokaryotic NAD‐IDHs are dimeric, such as NAD‐IDH from Acidithiobacillus thiooxidans (10) and Streptococcus mutans (11). In eukaryotes, mitochondrially localized NAD‐IDHs generate NADH to provide electrons for energy production (ATP), meanwhile NADP‐IDHs provide the reducing power (NADPH) and carbon skeleton (α‐ketoglutarate) for biosynthesis, cellular defense against oxidative damage and reactive oxygen species detoxification (12–15).…”