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

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 Mg2+ and Mn2+, respectively. The maximal activity for BlIDH occurred at 60 degrees C (with Mn2+) and 65 degrees C (with Mg2+), and pH 7.5 (with Mn2+) and pH 8.0 (with Mg2+). Heat-inactivation profiles revealed that BlIDH retained 50% of maximal activity after incubation at 45 degrees C for 20 min with either Mn2+ or Mg2+. 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.