Preparation And Functional Identification Of The Catalytic Subunit Of Acetohydroxyacid Synthase In Recombinant E.coli

Aacetohydroxyacid synthase?AHAS?is a rate-limiting enzyme that catalyzes the first common step in the branched-chain amino acid biosynthetic pathway.Since it is mainly present in plants,bacteria,and fungi,but absent in humans,it has been used as a potent drug targets for the screening of herbicides and antimicrobial drugs.AHAS of bacterial origin is composed of catalytic subunit?CSU?and regulatory subunit?RSU?.CSU also has catalytic activity when it is characterized alone;so many studies only prepared CSU for facility.Up to date,a plenty of studies have been carried out to elucidate the catalytic mechanism of AHAS,but because there is not a good way to simultaneously verify the comsumption of the substrate?s?and the product-spectrum in the reaction catalyzed by AHAS,especially in the reaction which implicates two substrates,namely pyruvate and 2-ketobutylrate.Therefore,there are still many undisclosed details concerning the catalytic mode of action of AHAS.To overcome the drawbacks of the methods currently employed in the determination of AHAS activity,we studied AHAS in this thesis by utilizing a pre-column derivatization-HPLC method which uses 4-nitro-o-phenylenediamine?NPDA?and 2,4-dinitrophenylhydrazine?DNPH?as derivatization reagents.Firstly,the cloning and expression of the CSU of E.coli AHAS I?Ec AHAS I-CSU?were performed.The recombinant bacterial strain which was named E.coli BL21?DE3?-p ET-28a?+?-ilv BN was successfully reconstructed and then Ec AHAS I-CSU was prepared in much better activity and stability.The specific activity of the protein is 12?mol?min^-1?mg^-1.Compared with the freshly purified protein,its activity reduced only by 30%when it was stored at 4?for two months;Furthermore,its activity did not significantly decrease after it had been stored at-80?for 4 months.Secondly,the reaction catalyzed by the enzyme with only pyruvate as the single substrate was analyzed by using both the traditional UV protocol and the pre-column derivatization-HPLC method mentioned above.The results of the UV method show that under such a condition,the yield of acetoin was about 80.2%and K_m^Pyr=6.57 m M,while the pre-column derivatization-HPLC method gave the yield of acetoin was 80.8%,the yield of 2,3-butanedione was about 18.4%,the consumption of pyruvate was around 99.6%,and K_m^Pyr=4.94 m M.From these data we can deduce that the latter method is more accurate in the evaluation of the reaction mediated by AHAS.Supported by these determinations,we evaluated the AHAS reaction involving dual substrates by the pre-column derivatization-HPLC method.The results show that the consumptions of the substrates pyruvate and 2-ketobutylrate are about99.72%and 98.22%,respectively and the products and their yields of are as follows:2,3-butanedione 2.44%,2,3-pentanedione 5.76%,acetoin 9.56%,2-hydroxy-3-pentanone10.38%,3-hydroxy-2-pentanone 64.1%,4-hydroxy-3-hexanone 4.8%,3,4-hexanedione 0.6%,acetaldehyde 0.5%,and propionaldehyde 0.84%,respectively.In this thesis,we comprehensively analyzed the substrate consumption and the product formation and composition of the reaction catalyzed by Ec AHAS I-CSU and their respective accurate contents for the first time,which provides a good foundation for the study of the mechanism of Ec AHAS I catalysis.Meanwhile,this research also confirms the speculations about the products in the reaction catalyzed by AHAS and deepens our understanding on the product spectrum of AHAS.More importantly,this study provides a valuable reference for the further research on the catalytic mechanisms of various AHAS obtaioned from different sources.