Characterization Of Type Ⅱ MEP Synthase And The Preliminary Exploration Of It’s Catalytic Mechanism

Background:The everlasting rise of antibiotic resistance has gradually invalidated some treatment of infection diseases,thus there is an urgent demand for novel antibiotics.The key enzyme of the MEP isoprenoid biosynthetic pathway named 1-deoxy-D-xylulose-5-phosphate reductoisomerase(DXR),is currently the most promising target for screening of novel antibiotics.DXR can be effectively inhibited by antibiotic fosmidomycin,which is used to treat multidrug resistant bacteria and malaria parasite.Recent research has shown that a few pathogenic bacteria including the pathogens Bartonella and Brucella do not have DXR but have an unrelated enzyme called DXR-like enzyme(DRL)or type Ⅱ DXR(DXR-Ⅱ).DRL and DXR differ phylogenetically and structurally despite catalyzing the same biochemical reaction,which infers that antibacterial drugs could be specifically designed against DRL.Method:We first sucessfully constructed E.coli BL21(DE3)-BaDRL strain and induced it with IPTG to express BaDRL.However,BaDRL thus produced showed low yield and unfavorable enymatic activity.The problem has not been overcome until the self-induction strategy was used.Moreover,through imitating glucolysis pathway in vitro,we used a"one pot" method to synthesize DXP,the substrate of DRL for research of the catalytic mechanism of BaDRL.Finally,we prepared[180-]MEP in H218O and by analyzing the reaction procedures and the labeling patterns and the isotope abundance of 0-18 labeled MEP,we provided more evidence for the catalytic mechanism of BaDRL.Result:We acquired enzyme BaDRL in high yield,good purity and with satisfactory activity by utilizing the self-induction method.Its optimal pH range is from 7 to 9 and it performs perfect at tempreature40 ℃.In addition,we determined the kinetic parameters of the enzyme.The results of 18O labeled experiments illustrated that the binding model between BaDRL and DXP is more likely in a C3-C4 binding manner.