The Enzymatic Properties And Detection Of Catalytic Reaction Products Of Bacteria-derived Ketol-acid Isomeroreductase

Ketol-acid isomeroreductase(KARI),also known as acetohydroxyacid isomeroreductase(AHIR),is one of the key enzymes in the branched-chain amino acid biosynthetic pathway.The three branched-chain amino acids(valine,leucine,and isoleucine)are essential amino acids which cannot be synthesized by human beings and must be obtained through food.However,bacteria,fungi,and plants can synthesize these compounds by themselves,so the key enzymes in the biosynthetic pathway of branching-chain amino acids can be used as potential targets for screening antimicrobial agents and herbicides.Therefore,it is of great significance to study the properties of KARI and the intermediary of reaction.There are two types of KARI enzymes.Mycobacterium tuberculosis KARI belongs to type I,which is composed of about 340 amino acids.Compared with type I,type II protein has a higher molecular weight and is composed of about 490 amino acids.This type of KARI is found in some bacteria,including Escherichia coli,and in all plants.The enzyme converts2-acetolactate(AL)or 2-acetyl-2-hydroxybutyrate(AHB)into their respective products,namely 2,3-dihydr-oxy-3-methylbutyrate and 2,3-dihydroxy-3-methyl-pentanate in a two-step sequential reaction.In this study,KARIs originated from M.tuberculosis and E.coli were separately induced and expressed in the engineered E.coli strains,and then purified by affinity chromatography to obtain EcKARI and MtKARI with high concentration and good purity.Since KARI needs to use NADPH as a coenzyme in the reaction,the enzyme activity can therefore be detected according to the consumption of NADPH in the enzymatic reaction using UV at 340 nm.The results of activity determination show that the consumption rate of substrate reaches more than 85%after being catalyzed by EcKARI or MtKARI,indicating that the two KARIs have high catalytic activity.On this basis,the enzymatic properties of the two enzymes were analyzed.The results show that the optimal reaction temperature of EcKARI and MtKARI are 37?,and the optimal p H are 7.0.In terms of metal ion dependence,the two proteins exhibit different characteristics.MtKARI could fully exert its catalytic activity only when Mg²⁺was used in the catalytic reaction,while EcKARI displays very high activity in the presence of Mn²⁺or Zn²⁺as well in addition to the use of Mg²⁺.At the same time,AL and AHB were used as substrates to measure the steady-state kinetic parameters of the two KARIs,and the results of AL are basically consistent with the data reported in previous papers.The results of AHB that were measured for the first time in this study are as follows:1)for EcKARI,K_m,AHB=0.58 m M,K_m,NADPH=0.085 m M;2)for MtKARI,K_m,AHB=0.46m M,K_m,NADPH=0.071m M.The products of KARI catalytic reaction are 2,3-dihydroxycarboxylic acid which have no chromogenic group and are difficult to be detected directly by ultraviolet or fluorescence detector.In this paper,the reaction products were decarboxylated,and then detected by HPLC or gas chromatography according to the physicochemical properties of the compounds after decarboxylation.A variety of experimental conditions have been tested,but no ideal results are obtained.This may be because the 2,3-dihydroxycarboxylic acids are difficult to be effectively decarboxylated under the selected experimental conditions.In the mean time,the determination of the compound 3-hydroxy-3-methyl-2-butyrate(HMOB)that has been considered as an intermediate in the KARI reaction and has never been directly detected in any experiments was initially explored by a precolumn derivatization-HPLC method in this thesis.Although the intermediate was yet not detectable,we made a reasonable speculation,that is in the reaction catalyzed by KARI,HMOB and the enzyme may form a tightly bound complex that makes it difficult to be released.The results of a series of studies on EcKARI and MtKARI carried out in this thesis provide a useful reference not only for further elucidation of the catalytic mechanism of KARI,but also for reasonable screening of antibacterial lead compounds using this enzyme as a target.