Enzymatic Characterization And Coenzyme Specificity Alteration Of Isocitrate Dehydrogenases From Bacillus Subtilis

Isocitrate dehydrogenase (IDH) is one of the key enzymes in the TCA cycle, which involves in providing energy and biosynthetic precursors for metabolism. Based on coenzyme specificity, the IDH family can be divided into the NADP⁺-dependent IDH (EC 1.1.1.42, NADP-IDH) and the NAD⁺-dependent IDH (EC 1.1.1.41, NAD-IDH). NADP-IDH is further classified into homodimeric and monomeric forms. The homodimeric NADP-IDH has been found in all of eukaryotes and most of prokaryotes, whereas monomeric NADP-IDH has been only found in a few of bacterial species.The isocitrate dehydrogenase from Bacillus subtilis (BsIDH), the wild-type isocitrate dehydrogenase from Escherichia coli (EcIDH) and its engineered mutant isocitrate dehydrogenase (EmIDH) are homodimeric IDHs and their enzymatic properties have not been determined well. In this thesis, these three kinds of IDHs were overexpressed and purified, and their enzymatic characterization was carried out in detail. The comprehensive information of enzymatic properties may provide the further insight into the catalytic mechanism of isocitrate dehydrogenase.Kinetics study showed that coenzyme specificities (calculated by the specific value of kcat/Km for NADP⁺ and NAD⁺) of BsIDH and EcIDH were 1330-fold and 3890-fold greater for NADP⁺ than NAD⁺, respectively. The mutant EmIDH showed 122-fold preference for NAD⁺ over NADP⁺. Both of BsIDH and EcIDH were typical NADP⁺-dependent IDHs, whereas EmIDH had been changed to be a NAD⁺-dependent enzyme. The Km values for isocitrate of BsIDH, EcIDH and EmIDH were 60.6μM, 67.6μM and 105.6μM, respectively. The optimal pH values of BsIDH, EcIDH and EmIDH were pH 8.2, pH 8.0 and pH 7.0, and their optimal temperatures were 45°C, 45°C and 43°C, respectively. The activity of three IDHs was entirely dependent on the binding of a divalent metal ion. The maximum activity of IDHs was observed in the presence of Mn²⁺ or Mg²⁺. The activity of IDHs was dramatically inhibited by other metal ions, such as Cu²⁺, Ca²⁺, Zn²⁺ and Ni²⁺. In order to convert the coenzyme specificity of BsIDH, the amino acid sequences of BsIDH and other IDHs from various bacteria species were aligned and analyzed, and seven amino acids were chosen as the mutant sites in BsIDH. Four mutantions (T3, T5, T6, T7) were constructed using site-directed mutagenesis method, and their kinetics were characterized. The result showed that the coenzyme specificity of mutant BsIDH was successfully inverted from NADP⁺ to NAD⁺. The T7 mutant enzyme displayed a 280-fold preference of NAD⁺ over NADP⁺. The crucial amino acids for the determination of cofactor specificity in EcIDH were also effective for the determination of cofactor specificity in BsIDH. Therefore, the cofactor specificity alteration of BsIDH would provide a basic evidence for the generality of the adaptive evolution mechanism of isocitrate dehydrogenases.