Enzymatic Characterization And Coenzyme Specificity Alteration Of Isocitrate Dehydrogenases From Xanthomonas Campestris. Campestris8004

Isocitrate dehydrogenase is a key enzyme in the tricarboxylic acid cycle （TCAcycle） that catalyzes the oxidative decarboxylation of isocitrate to2-oxoglutarate,CO₂and NAD（P）H, using NAD⁺or NADP+as coenzyme. The IDH catalysisprovides energy and biosynthetic precursors for cellular metabolism. Two types ofIDHs, NAD-specific IDH （EC1.1.1.41） and NADP-specific IDH （EC1.1.1.42）,can be divided according to their coenzyme specificity. NADP-IDH can beclassified into homodimeric and monomeric forms. Eukaryotes and mostprokarytotes have a homodimeric NADP-IDH, whereas monomeric NADP-IDHhas been found only in some bacteria. NAD-IDH can be divided into homodimericIDH and heterologous polymersomes IDH. Some prokaryotes and the archaeonpossess NAD-dependent homodimeric IDH.The gene encoding isocitrate dehydrogenase of Xanthomonas campestriscampestris8004（XcIDH） was cloned and overexpressed in Escherichia coliRosetta （DE3）. SDS-PAGE showed that purified XcIDH gave a single band （35kDa） and the native molecular mass was estimated to be61.9kDa by gel filtrationchromatography, suggesting that XcIDH is a homodimeric structure in solution.The activity of XcIDH was absolutely dependent on Mg²⁺and Mn²⁺, but wasstrongly inhibited by Zn²⁺and Ca²⁺. The optimal pH of XcIDH was pH7.5(Mn²⁺)and pH8.5(Mg²⁺), and the maximum activity was around45°C (Mn²⁺) and40°C(Mg²⁺), respectively. Heat inactivation study showed that XcIDH retained50%activity after20min of incubation at45°C in the presence of Mn²⁺. In addition, thekinetic study showed that the apparent Kmvalues were133μM for NAD⁺and3260μM for NADP+, using Mn²⁺as divalent cation. The enzyme performed a70-foldgreat specificity ([kN_cat/K_m]AD/[k_cat/KDP+m]^NA) for NAD⁺than NADP.In order to further study the molecular mechanism of coenzyme specificity of IDH, the coenzyme specificity of XcIDH was converted in this study. The aminoacid sequences alignments showed that the Asp268、Ile269and Ala275were thekey residuals binding the cofactor NAD⁺. These residuals were replaced withLys268、Tyr269and Val275by site-directed mutagenesis. The result showed thatthe mutant XcIDH performed a17-fold great specificity coenzyme([kDPcat/Km]^NA/[kN_cat/K_m]^AD) for NADP⁺than NAD⁺, which proved specificitycoenzyme of mutant XcIDH was successfully converted from NAD⁺to NADP⁺.The previous investigation focused on the enzymatic characterization andcrystal structure of NADP-IDH, while the report about the enzymatic property andevolutional relationship of NAD-IDH was limited. This study provides the firstexperimental evidence that XcIDH is a divalent cation-dependent homodimericIDH, which displays remarkably high thermostability. All those studies willprovide useful information for secondary metabolite utilizations of X. campestris.