Functional Investigation Of A Novel NAD~+-Dependent Monomeric Isocitrate Dehydrogenase

Isocitrate dehydrogenase?IDH?is a key enzyme in the tricarboxylic acid?TCA?cycle,which plays an important role in energy metabolism and anabolism as a housekeeping protein.IDH catalyzes the oxidative decarboxylation of isocitrate to yield ?-oxoglutarate and CO₂ coupled with the reduction of NAD?P?⁺.Based on the coenzyme specificity,IDH can be divided into NAD⁺ dependent IDH?NAD-IDH,EC 1.1.1.41?and NADP⁺ dependent IDH?NADP-IDH,EC 1.1.1.42?.According to the subunit structure,IDH falls into 4 types,monomeric IDH,homodimeric IDH,homotetrameric IDH and hetero-oligomeric IDH.Phylogenetic analysis showed that NAD⁺ use is an ancestral trait of IDH and NADP⁺ use is an adaptive phenotype,which has been proved for Type I subfamily in the laboratory.However,all reported monomeric IDHs are NADP⁺-dependent enzymes.Therefore,the ancestor of monomeric NADP-IDH is still unclear.In this study,the phylogenetic tree was reconstructed,and IDH family can be classified into three subfamilies,Type I IDH subfamily,Type II IDH subfamily and monomeric IDH subfamily.It was found that there are two distinct clades in the monomeric subfamily,one is monomeric NADP-IDH and the other contains IDHs from Campylobacter which forms a new clade.Sequence alignment illustrated that the key residues in the coenzyme-binding pocket of new IDH are very different from those in the typical monomeric NADP-IDH.The gene encoding an IDH from Campylobacter sp.FOBRC14 was synthesized and heterologously expressed in Escherichia coli.SDS-PAGE analysis showed that the molecular weight of purified CaIDH was about 81 kDa,and MALDI-TOF/TOF mass spectrometry analysis showed that the molecular weight was 81.9 kDa,suggesting a typical monomeric IDH.Mn²⁺ was the most preferred divalent cation for Ca IDH activity.In the presence of Mn²⁺ and Mg²⁺,the optimum pH of CaIDH were 7.5 and 8.0 and the optimum temperature were 40 °C and 45 °C,respectively.Thermal stability studies showed that CaIDH was stable below 45 °C,but its activity decreased rapidly over 45 °C.The specific activities of Ca IDH were 54.6 U/mg with NAD⁺ and only 11.3 U/mg with NADP⁺,respectively,which confirmed that CaIDH prefers NAD⁺ over NADP⁺.Kinetics showed that the K_m values of Ca IDH for NADP⁺ was 4.2-fold(Mn²⁺)and 17.8-fold(Mg²⁺)over NAD⁺,respectively.Thus,the affinity of CaIDH for NAD⁺ was much higher than that for NADP⁺.The coenzyme preference of CaIDH for NAD⁺((k_cat/K_m)^NAD/(k_cat/K_m)^NADP)was 19.4-fold(Mn²⁺)and 61-fold(Mg²⁺)over that for NADP⁺,respectively.Evidently,Ca IDH is a novel NAD⁺-dependent monomeric IDH.To evaluate the significance of the putative coenzyme determinant sites?Leu584 and Asp595?,double-point mutant enzyme(CaH⁵⁸⁴R⁵⁹⁵)was constructed successfully.Circular dichroism analysis indicated that the mutations did not cause significant changes in protein secondary structure.The K_m values of mutant CaH⁵⁸⁴R⁵⁹⁵ for NADP⁺ were 76.5 ?M(Mn²⁺)and 11.4 ?M(Mg²⁺),respectively.As compared with the wild-type enzyme,the affinities of CaH⁵⁸⁴R⁵⁹⁵ toward NADP⁺ were increased by 9.7-fold(Mn²⁺)and 45-fold(Mg²⁺)and the catalytic efficiencies were increased by 27-fold(Mn²⁺)and 92-fold(Mg²⁺),respectively.However,the affinity and catalytic efficiency toward NAD⁺ was significantly reduced.The coenzyme preference of CaH⁵⁸⁴R⁵⁹⁵ toward NADP⁺((k_cat/K_m)^NADP/(k_cat/K_m)^NAD)was 14.5-fold(Mn²⁺)and 19-fold(Mg²⁺)over that for NAD⁺,respectively,indicating that the coenzyme specificity of CaIDH has been converted from NAD⁺ into NADP⁺ successfully.In the current study,the icd gene?encoding IDH?on the chromosome of E.coli MG1655 was replaced by genes encoding CaIDH and CaH⁵⁸⁴R⁵⁹⁵ through homologous recombination,creating two mutated strains(MG::CaIDH and MG::CaH⁵⁸⁴R⁵⁹⁵).The growth rates of four strains,MG::CaIDH,MG::CaH⁵⁸⁴R⁵⁹⁵,the wild-type strain?E.coli WT?containing NADP⁺-dependent E.coli IDH?Ec IDH?and mutated E.coli Ym containing engineered NAD⁺-dependent EcIDH,were determined in the different media.During growth on glucose,the growth rate of the mutant strain,MG::CaH⁵⁸⁴R⁵⁹⁵,was 82% of that of the wild-type strain,whereas the other three strains had similar growth rate.During growth on glucose,three mutant strains had lower growth rate as compared with the wild-type strain.MG::CaH⁵⁸⁴R⁵⁹⁵ grew very poorly,and its growth rate was only 65.5% of that of E.coli WT,which may be caused by its poor activity.Although it has not been found that the activity of any monomeric IDH is regulated by phosphorylation as dimeric EcIDH,the monomeric IDH may play similar physiological roles as dimeric IDH.The further analysis of the metabolic pathways in four strains is in progress using omics techniques.The discovery of the novel monomeric NAD-IDH is an important complement to the phylogeny of IDH family,and provides the fundamental information for exploring the molecular evolution mechanism of monomeric IDH.