Mechanisms of Catalytic Function of Prenyl Chain Elongating Enzymes

1)十一碳烯基二磷酸合成酶基因的克隆和Z-戊烯基链延长酶保守序列的鉴定通过建立一种新的筛选方法来检测在大肠杆菌细胞中高表达额外戊烯基转移酶的菌落，从而为催化Z-戊烯基链延长的戊烯基转移酶提供第一一级结构，从而克隆了十一碳烯基二磷酸合成酶(UPS)基因。推测的UPS一级结构与E-戊烯基链延长酶的一级结构完全不同，后者具有特征的保守区，包括富含天冬氨酸的基序。成功构建了使UPS在E.COIL细胞中高效表达的基因表达系统。2)枯草杆菌七烯基二磷酸合成酶功能表达机制的研究枯草芽孢杆菌的七烯基二磷酸合成酶由t-…组成更多的两个可分离的亚基、组分和I-II，这两个都是催化功能所必需的。通过凝胶过滤和免疫印迹分析研究了催化活性络合物形成过程中的动态相互作用。通过假设这两个亚基的结合和解离有助于催化从可溶底物中合成两亲性水不溶产物，从而理解了这种异构酶的作用机制。利用烯丙基底物的苯甲酰苯氧基类似物法尼基二磷酸(FPP)进行的光亲和标记实验表明，组分I通过疏水的戊烯基尾参与FPP的结合，两个组分与烯丙基底物形成稳定的三元络合物。3)通过基因工程引入硬脂嗜热芽孢杆菌FPP合成酶结构基因的多种突变，对枯草杆菌HEPPP合成酶和枯草芽孢杆菌的UPS进行鉴定，寻找对催化功能或底物结合重要的氨基酸残基。较少

1) Molecular Cloning of Undecaprenyl Diphosphate Synthase Gene and Identification of Conserved Sequences for Z-Prenyl Chain Elongating EnzymesCloning of the gene for undecaprenyl diphosphate synthase (UPS) was carried out by developing a novel screening method to detect colonies overexpressing extra prenyltransferases in Escherichia coli cells by autoradiography, providing the first primary structure for any prenyltransferases that catalyze Z-prenyl chain elongation. The deduced primary structure of the UPS is totally different from those of E-prenyl chain elongating enzymes which have characteristic conserved regions including aspartate-rich motifs. Construction of the gene expression system which enabled overproduction of the UPS in E.coil cells was successful. Purification and crystallization of the UPS was carried out.2) Elucidation of the Function-Expressing Mechanisms of Heptaprenyl Diphosphate SynthaseHeptaprenyl diphosphate synthase (HepPS) of Bacillus subtilis is composed of t … More wo dissociable subunits, componentsand I -II, both of which are required for the catalytic function. The dynamic interaction in forming a catalytically active complex was investigated by gel filtration and immunoblotting analysis. The mechanism of this heteromeric enzyme is understood by assuming that association and dissociation of the two subunits facilitate turnover of catalysis for the synthesis of the amphipathic water-insoluble product from soluble substrates. Photoaffinity labelling experiments by use of a benzoylphenoxy analog of the allylic substrate, farnesyl diphosphate (FPP) indicated that component I participates in binding of FPP through the hydrophobic prenyl tail and that the two components with the allylic substrate form a stable ternary complex.3) Identification of Active Sites of Prenyltransferases by Genetic Engineering Introduction of many kinds of mutations in the structural genes of FPP synthase of Bacillus stearothermophilus, HepPP synthase of B.subtilis and UPS of M.Iuteus B-P 26 was carried out to find amino acid residues that were important for thecatalyticfunction or substrate binding. Less