Functional Investigation Of Isocitrate Dehydrogenases From Acinetobacter Baumannii And Saccharomyces Cerevisiae

Isocitrate dehydrogenase(IDH)is the key enzyme in the tricarboxylic acid cycle(TCA),which catalyzes the oxidative decarboxylation of isocitrate to produce?-ketoglutarate(?-KG),reduced coenzyme NAD(P)H,CO₂ and ATP.IDH,an important housekeeping protein distributed in all living organisms,plays essential roles in catalysis and regulation in life.1.Functional and structural investigation of two kinds of isocitrate dehydrogenases from Acinetobacter baumanniiAcinetobacter baumannii is a gram-negative opportunistic pathogen.Currently,it is an important nosocomial infection pathogen around the world.Two open reading frames ofIDH genes are found in the genome of A.baumannii,encoding AbIDH1 composed of418 amino acids and AbIDH2 composed of 745 amino acids,respectively.In this study,the biochemical characteristics of AbIDH1 and AbIDH2,the potential phosphorylation of Ser113 in AbIDH1 and the crystal structure of AbIDH2 were investigated.This work will provide new ideas for the relationship betweenIDH function and the pathogenicity of A.baumannii,and the evolutionary relationship between the dimeric and monomericIDHs.(1)Characterization of enzymology and potential phosphorylation site of AbIDH1AbIDH1 gene from A.baumannii was cloned and heterologously expressed in E.coli Rosetta(DE3).The molecular mass of purified AbIDH1 was 83.5 kDa determined by gel filtration chromatography,suggesting a homodimeric structure.Both metal ions,Mg²⁺and Mn²⁺,were the best activators for AbIDH1.Co²⁺,Ni²⁺and Ca²⁺significantly inhibited the activity of AbIDH1.The maximal activity was found at 45°C,and the optimum p H was 8.2 with Mg²⁺and 7.5 with Mn²⁺.AbIDH1 was a NADP⁺-specific enzyme,and its specific activities were 39.8 U/mg and 59.8 U/mg in the presence of Mg²⁺and Mn²⁺,respectively.Kinetics showed that the apparent K_m values of AbIDH1 for isocitrate were68.9?M with Mg²⁺and 50.5?M with Mn²⁺,and the apparent K_m values for NADP⁺were18.1?M with Mg²⁺and 46.6?M with Mn²⁺,respectively.The phosphorylation experiment in vitro illustrated that AbIDH1 can be phosphorylated byIDH kinase/phosphatase of E.coli.The mass spectrometry analysis showed that Ser113 and Ser22 were the phosphorylated sites in AbIDH1.To identify the functions of Ser113 and Ser22,nine mutant enzymes were constructed.The enzyme assay revealed that S113E,S113D and S113Y totally lost the activity.The activities of S113A,S113T and S113G were also remarkably decreased.However,the S22E,S22H and S22A mutations had no influence at all.Therefore,the conserved Ser113 is the potential phosphorylation site of AbIDH1.(2)Enzymatic characterization,and structure elucidation and engineering of AbIDH2AbIDH2 gene was cloned and heterologously expressed in E.coli Rosetta(DE3).The molecular mass of purified AbIDH2 was 189 kDa and 156 kDa,respectively,determined by gel filtration chromatography and ultracentrifugation,suggesting a homodimeric structure.Mn²⁺was the most effective activator of AbIDH2.Co²⁺and Ni²⁺had no influence on AbIDH2 activity,which was different from AbIDH1.In the presence of Mn²⁺,AbIDH2 showed the maximal activity at 45°C,pH 7.2.Heat inactivation studies revealed that the activity of AbIDH2 was lost rapidly above 42°C.AbIDH2 was also a NADP⁺-specific enzyme,and its specific activity was 25.4 U/mg with Mn²⁺.Kinetics showed that the apparent K_m values of AbIDH2 for isocitrate and NADP⁺were 20.6?M and 93.7?M,and the k_cat of AbIDH2 for isocitrate and NADP⁺were 39.2 s^-1 and 36.9 s^-1,respectively.The phylogenetic analysis reveals thatIDH family can be divided into three subfamilies,Type ? subfamily,Type ? subfamily and Type ? subfamily.AbIDH2 is a member of Type ? subfamily,which is a novel homodimericIDH and shares high sequence similarity with the monomeric NADP-IDH.The crystal structure at 3.0(?)resolution reveals that two subunits of homodimeric AbIDH2 interact with each other through salt bonds,hydrogen bonds and other non-bonded interaction.In this study,the homodimeric AbIDH2 was converted into a monomeric enzyme(mono-AbIDH2)by engineering the interface of homodimer with seven residue mutations.It was observed that mono-AbIDH2 had similar catalytic efficiency to the homodimeric AbIDH2.According to the analysis of protein 3D structure comparison,the topological structure of AbIDH2 was very similar to the homodimeric NADP-IDH of Type ? subfamily,whereas the topological structure of AbIDH2 single subunit was significantly similar to the monomeric NADP-IDH of Type ? subfamily.Furthermore,the affinity of AbIDH2for substrate was higher than dimeric AbIDH1 in Type ? subfamily,but much lower than the monomericIDH in Type ? subfamily.Also,the catalytic efficiency of AbIDH2 was slightly higher than dimeric AbIDH1,but much lower than the monomericIDH.Taken together,it is proposed that the dimeric NADP-IDH in Type ? subfamily with AbIDH2 as a typical member may be the undiscovered intermediate forms in the evolutionary relationship between dimericIDH and monomericIDH.2.Functional investigation of Arg148 in from Saccharomyces cerevisiaeIDH1R132 mutations in human cytoplasmIDH1(HcIDH1)are involved in the tumorigenesis.IDH R132 mutation lost the function in catalyzing the oxidation of isocitrate,but acquired a neomorphic function in catalyzing the reduction of?-KG.Arg148is the homologous residue to R132 in the mitochondrial NADP⁺-dependentIDH1 from Saccharomyces cerevisiae(ScIDH1).Three ScIDH1 mutant enzymes,R148H,R148A and R148E,were constructed by site-directed mutagenesis.As compared with the catalytic efficiency of the wild-type ScIDH1,that of R148H,R148A and R148E was decreased by227-fold,215-fold and 500-fold,respectively.Although ScIDH1 activity was dramatically reduced by R148 mutation,the mutant enzymes had neomorphic function in catalyzing the reduction of?-KG.The reduced product of?-KG was identified to be 2'-hydroxyglutarate(2'-HG),an oncometabolite,by GC/TOF-MS.Therefore,ScIDH1 R148H mutant has similar biological function to HcIDH1 R132H mutant.A yeast model Sc::R148H was constructed to explore the effects of R148H mutation on the yeast metabolism.i TRAQ-based proteomics analysis revealed that 75 proteins showed differential expression between the wild-type yeast and mutant Sc::R148H,in a total of 3072 proteins.42 proteins were up-regulated and 33 proteins were down-regulated.In the future,the further investigation of yeast proteomes will provide a new insight into the relationship betweenIDH mutations and the molecular mechanism of tumorigenesis.