Enzymatic Characterization And Function Engineering Of Two Structurally Different Isocitrate Dehydrogenases From Xylella Fastidiosa

Isocitrate dehydrogenase?IDH?is a crucial enzyme that locates at a critical junction between the tricarboxylic acid cycle and the glyoxylate bypass,thus controlling the carbon flow between these two pathways.Isocitrate lyase?ICL?is a key enzyme of glyoxylate shunt and usually coexists with an NADP-IDH in bacteria.Most bacteria have only one IDH,while a small number of bacteria contain two IDH isoenzymes.The cause of the coexistence of two different IDHs in one organism was unclear.Xylella fastidiosa is a nutritionally fastidious plant pathogen that contains two structurally different IDHs,an NAD⁺-dependent homodimeric IDH?diXfIDH?and an NADP⁺-dependent monomeric IDH?monoXfIDH?.Interestingly,Xylella fastidiosa has no sequence homologous to ICL gene in its genome,although it has an NADP⁺-dependent monomeric IDH.Therefore,to investigate the biochemical properties of these two interesting IDHs was valuable.In this study,two wild-type enzymes,diXfIDH and monoXfIDH,and 5 mutant enzymes were heteroexpressed,purified and determined.SDS-PAGE analysis showed that the subunit molecular masses of diXfIDH and monoXfIDH were around 38 kDa and82 kDa,respectively,which were consistent with the theoretical values.However,the native molecular weights of diXfIDH and monoXfIDH were estimated to be about 59kDa and 76 kDa by size exclusion chromatography?SEC?,respectively,which were lower than the theoretical value of diXfIDH?76 kDa?and monoXfIDH?82 kDa?.It may be due to a compact packing structure of two subunits or non-ideal interactions between the protein and the SEC media.Then,the MALDI-TOF/TOF mass spectrometry was performed and the molecular masses of diXfIDH and monoXfIDH were precisely determined to be 75.5 kDa and 82.4 kDa,respectively.Circular dichroism spectroscopy showed very similar profiles for the wild-type and mutant enzymes,suggesting that the protein secondary structure had no evident changes by a few amino acid substitutions.Kinetics studies showed that diXfIDH displayed 206-fold preference for NAD⁺over NADP⁺,while monoXfIDH showed 13785-fold preference for NADP⁺over NAD⁺.The optimum pH for diXfIDH was between 8.0 and 9.0,while the optimum pH for monoXfIDH was 7.75.The optimum temperature for diXfIDH and monoXfIDH was55°C and 50°C,respectively.The heat inactivation studies showed that diXfIDH was stable below 40°C,but lost half of the original activity after incubation for 20 min at47°C.The optimum temperature for monoXfIDH was 50°C.However,the thermal stability of monoXfIDH was poor as compared with diXfIDH.MonoXfIDH was stable below 35°C,but its activity decreased rapidly at temperatures above 35°C.This enzyme became almost denatured after incubation at 42.5°C for 20 min.Mg²⁺was the most favored divalent cation for diXfIDH,while Mn²⁺was the most favored ion for monoXfIDH.DiXfIDH activity was completely inhibited by Cu²⁺and Zn²⁺and monoXfIDH activity was inhibited by Zn²⁺.The putative crucial amino acids(D²⁶⁸,I²⁶⁹ and A²⁷⁵ in diXfIDH and K⁵⁸⁹,H⁵⁹⁰ and R⁶⁰¹ in monoXfIDH)for coenzyme binding were studied by site-directed mutagenesis.The preference of three diXfIDH mutants?D268K,D268K/I269Y and D268K/I269Y/A275V?for NADP⁺were 3-,40-,and 62-fold over NAD⁺,respectively,demonstrating that the coenzyme specificities of these mutants were switched from NAD⁺to NADP⁺successfully.Meanwhile,the affinity of two monoXfIDH mutants?H590L/R601L and K589T/H590L/R601L?for NADP⁺were decreased by 1506-fold and 959-fold,respectively.However,the mutations failed to improve the activity of mutant enzymes using NAD⁺as a coenzyme.The monoXfIDH mutants became the enzymes with dual coenzyme specificities.In the current study,two structurally different IDHs?diXfIDH and monoXfIDH?from X.fastidiosa were identified in details.Both of diXfIDH and monoXfIDH were high-efficiency enzymes.Surprisingly,monoXfIDH has the highest activity than all known monomeric counterparts.Besides,monoXfIDH is not coupled with ICL in X.fastidiosa.Therefore,this work would provide useful information for the further insight into the relationships among NADP-IDH,ICL and the glyoxylate bypass.