Biochemical Characterization and Complete Conversion of Coenzyme Specificity of Isocitrate Dehydrogenase from Bifidobacterium longum

Abstract

Bifidobacterium longum is a very important gram-positive non-pathogenic bacterium in the human gastrointestinal tract for keeping the digestive and immune system healthy. Isocitrate dehydrogenase (IDH) from B. longum (BlIDH), a novel member in Type II subfamily, was overexpressed, purified and biochemically characterized in detail. The active form of BlIDH was an 83-kDa homodimer. Kinetic analysis showed BlIDH was a NADP⁺-dependent IDH (NADP-IDH), with a 567- and 193-fold preference for NADP⁺ over NAD⁺ in the presence of Mg(2+) and Mn(2+), respectively. The maximal activity for BlIDH occurred at 60 °C (with Mn(2+)) and 65 °C (with Mg(2+)), and pH 7.5 (with Mn(2+)) and pH 8.0 (with Mg(2+)). Heat-inactivation profiles revealed that BlIDH retained 50% of maximal activity after incubation at 45 °C for 20 min with either Mn(2+) or Mg(2+). Furthermore, the coenzyme specificity of BlIDH can be completely reversed from NADP⁺ to NAD⁺ by a factor of 2387 by replacing six residues. This current work, the first report on the coenzyme specificity conversion of Type II NADP-IDHs, would provide better insight into the evolution of NADP⁺ use by the IDH family.

Keywords: Bifidobacterium longum; biochemical characterization; coenzyme specificity determinants; isocitrate dehydrogenase; kinetics.

Figure 1

Phylogenetic tree of 26 isocitrate dehydrogenases (IDHs). A neighbor-joining tree with 500 bootstrap was created using MEGA 6.06. The GenBank accession numbers were noted in the parentheses.

Figure 2

Structure-based protein sequences alignment of isocitrate dehydrogenase (IDH) from B. longum (BlIDH) with other IDHs. The primary residues involved in substrate binding are indicated by pentagrams (★). The residues interact with the 2′-phosphate of NADP⁺ directly or indirectly are indicated by triangles (▲). The letters in blue boxes indicate conserved residues, and the white letters with red background in blue boxes indicate strictly conserved residues. The black letters in white boxes indicate similarity. The structure of BlIDH was generated by SWISS-MODEL server. The figure created by ESPript 3.0.

Figure 3

Comparison of the NADP⁺-binding sites among the human cytosolic NADP-IDH (HcIDH), BlIDH, Acidithiobacillus thiooxidans NAD-IDH (AtIDH) and Ostreococcus tauri NAD-IDH (OtIDH). (A) Overlay of the subunits of HcIDH (yellow, PDB code: 1T0L), modelled BlIDH (cyan) and AtIDH (green, PDB code: 2D4V) highlighting the selected coenzyme binding sites in these three IDHs. The NADP⁺ molecule (with yellow C atoms) and NAD⁺ molecule (with green C atoms) were represented by the stick. The model of BlIDH was generated by SWISS-MODEL server; (B) A close-up view showing the selected residues involving in NADP⁺ binding in HcIDH (labelled by black). The equivalent residues in BlIDH, targeted by site-directed mutagenesis, and in OtIDH were labelled by red and blue, respectively.

Figure 4

Overexpression and molecular mass determination of BlIDH. (A) SDS-PAGE analysis. M, molecular mass marker; lane 1, crude extract from cells transformed by pET-28b(+) with IPTG treatment; lane 2, crude extract from cells transformed by recombinant plasmid pET-BlIDH with IPTG treatment; lane 3, purified BlIDH; (B) Western blot analysis. Lane 1, purified BlIDH; lane2, negative control, crude extract from cells transformed by pET-28b(+) with IPTG treatment; (C) Size exclusion chromatography (SEC) analysis of BlIDH.

Figure 5

Effect of pH and temperature on the activity of BlIDH in the presence of Mg²⁺ (●) and Mn²⁺ (■), respectively. (A) Effect of pH on the activity of BlIDH; (B) Effect of temperature on the activity of BlIDH; (C) Heat-inactivation profiles of the BlIDH. The values indicate the means of at least three independent measurements.