Studies On Key Amino Acid Residues Of 1-deoxy-D-xylulose-5-Phosphate Synthase(DXS)in E.coli

Terpenoids are a wide variety of natural compounds with multiple biological activities.Two routes,namely the mevalonate(MVA)pathway and the 2-methyl-D-erythritol-4-phosphate(MEP)pathway have been elucidated for the biosynthesis of terpenoids.Because the MEP pathway does not exist in mammals,and is only found in most bacteria,including pathogenic bacteria,this pathway has become a promising targets for new antibiotics,antimalarials,and herbicides.1-Deoxy-D-xylulose-5-phosphate synthase(DXS),the first key rate-limiting enzyme in the MEP pathway,catalyzes the condensation reaction between pyruvate and D-glyceraldehyde-3-phosphate(D-GAP)to form 1-deoxy-D-xylulose 5-phosphate(DXP),In the following steps,DXP is catalyzed by a series of enzymes including 1-deoxy-D-xylulose-5-phosphate reductoisomerase(DXR)to generate terpenoid precursors isopentenyl pyrophosphate(IPP),and dimethyl allyl pyrophosphate(DMAPP).Therefore,the studies of the key amino acid residues of DXS are of particular importantThis thesis focuses on the key amino acid residues of E.coli DXS.Some researchers have found that the arginine residues at the positions 420 and 478 of E.coli DXS form a cationic pocket near the active site to accommodate the phosphate group of D-GAP,which plays a role of carbon transfer in the later stage of DXP formation.In addition,some researchers have shown that the 49th histidine residue of E.coli DXS is highly homologous in TPP-dependent enzymes,and the mutant EcDXS-H49Q has lost its biological activity.Based on the above,we used site-directed mutagenesis to obtain 10 mutants,including 5 double mutants based on EcDXS-H49Q,namely EcDXS-H49QR420D,EcDXS-H49QR420P,EcDXS-H49QR420F,EcDXS-H49QR478D,EcDXS-H49QR478P,and 5 single mutants:EcDXS-R420D,EcDXS-R420P,EcDXS-R420F,EcDXS-R478D,EcDXS-R478P.The enzyme coupling method was used to determine the activity of these mutants.The five double mutants and the single mutant EcDXS-R420P lost their catalytic activities.The results show that when a positively charged basic amino acid is mutated to a negatively charged acidic amino acid or a non-polar and benzene ring amino acid,the cationic pocket formed by amino acid residues at positions 420 and 478 is destroyed,making the phosphate group of D-GAP cannot tightly bind to DXS.However,the mutant EcDXS-R478P still retained 50%of its biological activity.The reason may be that after the positively charged Arg residue at position 478 has been mutated to non-polar Pro,although its interaction with the phosphate group of D-GAP becomes smaller,it is not as mutually exclusive as the negatively charged amino acid residues.Therefore,Ec-DXS-R478P still retains some activityFurther,we used a pre-column derivatization HPLC method to study the substrate spectrum of EcDXS in depth.Wt-EcDXS and the 10 EcDXS mutants were used to catalyze the reaction of D-GAP with pyruvate,nitrosobenzene with pyruvate and benzaldehyde with pyruvate.It is found that Wt-EcDXS,the mutants EcDXS-R420D,EcDXS-R420F.EcDXS-R478D,and EcDXS-R478P can all catalyze the above reactions to different extents.It is further speculated that the combination of nitrosobenzene and benzaldehyde with the enzyme during the reaction with pyruvate may have some consistency with that of D-GAP.In addition,we also found that both Wt-EcDXS and mutants EcDXS-R478D and EcDXS-R478P can catalyze the reaction of pyruvate itself and the reaction of pyruvate with 2-butanoic acid,but unfortunately no reaction product has been detected.Further research found that DXS still retains the natural properties of TPP-dependence in the process of catalyzing the reaction of pyruvate itself,but the cofactor Mg2+only promotes this reaction weakly.