Enzymatic Properties Of Enzymes Related To Branched-chain Amino Acid Biosynthetic Pathway Of Pseudomonas Syringae Pv.tomato DC3000

Acetohydroxyacid synthase（AHAS）and ketol-acid reductoisomerase（KARI）are two key enzymes in the BCAA biosynthetic pathway,whose catalysis and mechanism have received extensive attention.The holoenzyme of AHAS contains catalytic subunits（CSU）and regulatory subunits（RSU）,catalyzed production of（S）-2-acetyl lactate（two molecules of pyruvate as substrate）or（S）-2-acetyl-2-hydroxybutyrate（pyruvate and 2-ketobutyrate as substrate）,and following in the step,these two compounds are converted respectively to（2R）-2,3-dihydroxy-3-methylbutyrate（L-valine and L-leucine precursors）or（2R,3R）-2,3-dihydroxy-3-methylpentanate（L-isoleucine precursor）under KARI catalysis.Pseudomonas syringae pv.tomato DC3000（PstDC3000）can cause plant leaf chlorosis by secreting coronatine.Studies have found L-allo-isoleucine is a precursor of coronatine synthesis,but the biosynthetic pathway for this unusual amino acid remains yet determined.So we speculate based on the L-isoleucine biosynthetic pathway,that L-allo-isoleucine may be biosynthesized using 2-butanone acid（donor substrate）and pyruvate（acceptor substrate）catalyzed by AHAS to produce（S）-2-propanoyl lactate,compound may be transformed to L-allo-isoleucine by three consecutive reaction steps mediated respectively by KARI,dihydroxyacid dehydratase（DHAD）,and amino transferase（BCAT）.In this thesis,acetohydroxyacid synthase-CSU（PstAHAS-CSU）,PstAHAS-RSU,and ketol-acid reductoisomerase（PstKARI）derived from PstDC3000 with good purity were obtained by expression and purification.In addition,PstAHAS holoenzyme was prepared through in vitro reconstruction of PstAHAS-CSU and PstAHAS-RSU.Subsequently,the activity of PstAHAS-CSU and PstAHAS holoenzyme were determined by creatine-naphthol colorimetric method.The data showed that the activity of the holoenzyme was significantly improved compared to AHAS-CSU.On this basis,the fundamental enzymatic properties of PstAHAS-CSU were studied.The K_mvalue(K_m^Pyr=7.7 m M)of PstAHAS-CSU in its catalyzed reaction using pyruvate as a single substrate was detected under steady-state conditions.The feedback inhibition effect of BCAAs on the activity of PstAHAS-CSU and PstAHAS holoenzyme was further studied,and the results verified that BCAA affected the activity of AHAS holoenzyme by acting on the RSU,they had no effect on the activity of AHAS-CSU.Pre-column derivatization-HPLC method was used to analyze the consumption of the substrate and the formation of products in the PstAHAS-CSU and PstAHAS holoenzyme catalyzed reactions employing pyruvate/2-ketobutyrate as a single substrate or using pyruvate and 2-ketobutyrate as dual substrates.The results showed that in the dual substrate reactions,PstAHAS is more inclined to taking pyruvate as the donor substrate,but can also utilize 2-ketobutyrate as the donor substrate and pyruvate as the acceptor substrate to generate（S）-2-propionyllactate.This preliminarily clarifies our speculation on the biosynthetic pathway of L-allo-isoleucine.The research results of PstKARI in this thesis indicate that the conversion rate of PstKARI substrate reaches 71.2%,the optimal reaction temperature is 37°C,and the optimal reaction p H is 7.5（PB buffer）.In terms of the effect of metal ions on the enzyme activity,it was found that Mg²⁺is the best activator of enzyme activity,while other metal ions had different activation on the enzyme and Fe²⁺and Cu²⁺exhibit the lowest effect on KARI activity.At the same time,the K_mvalues of different substrates in PstKARI reactions were determined(K_m^AL=0.37 m M,K_m^AHB=0.28 m M).In this research,the catalysis of PstAHAS and PstKARI was analyzed initially,and the biosynthetic pathway of L-allo-isoleucine was preliminarily explored,which not only laid sound foundation for the later in-depth study of the biosynthesis of L-allo-isoleucine,but provided data and references for future investigations of different AHASs and KARIs.