YEAST FARNESYL-DIPHOSPHATE SYNTHASE - SITE-DIRECTED MUTAGENESIS OF RESIDUES IN HIGHLY CONSERVED PRENYLTRANSFERASE DOMAIN-I AND DOMAIN-II
YEAST FARNESYL-DIPHOSPHATE SYNTHASE - SITE-DIRECTED MUTAGENESIS OF RESIDUES IN HIGHLY CONSERVED PRENYLTRANSFERASE DOMAIN-I AND DOMAIN-II
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DOI：
10.1073/pnas.91.8.3044
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发表时间：
1994-04-12
影响因子：
11.1
通讯作者：
POULTER, CD
中科院分区：
文献类型：
--
作者：
SONG, LS;POULTER, CD
异戊烯基转移酶催化类异戊二烯生物合成途径中的基本链延伸反应，含有几个高度保守的氨基酸，包括两个富含谷氨酸的区域，被认为参与底物结合和催化。我们报告了酵母法呢基二磷酸合酶（FPPSase;香叶基二磷酸：异戊烯基二磷酸，EC 2.5.1.10）的定点突变体的研究，该酶是一种异戊烯基转移酶，催化异戊烯基二磷酸（IPP）与二甲基烯丙基二磷酸和香叶基二磷酸的顺序1 '-4偶联。通过C-末端-Glu-Glu-Phe α-微管蛋白表位（单字母氨基酸代码中的EEF）延伸的FPPSase的重组形式被工程化以促进通过免疫亲和色谱法快速纯化酶并从大肠杆菌宿主中的野生型FPPSase去除痕量的污染活性。在FPPSase：：EEF中构建了10个定点突变体。域I中的六个州（在位置100、101和104）和结构域II（在位置240、241和244）被改变为丙氨酸（突变体命名为D100 A、D101 A、D104 A、D240 A、D241 A和D244 A）;三个精氨酸残基发生变化，Arg-109和Arg-110变为谷氨酰胺，Arg-350变为丙氨酸（突变体命名为R109 Q、R110 Q和R350 A）;并且Lys-254转化为丙氨酸（突变体命名为K254 A）。结构域I中的组氨酸残基和附近的精氨酸残基以及结构域II中的Asp-240和Asp-241的突变显著降低了FPPSase：：EEF的催化活性。D244 A和K254 A突变体的活性显著降低，而R350 A突变体的k（cat）和米氏常数与FPPSase：：EEF的相似。在野生型FPPSase的C末端添加-EEF表位导致K（m）IPP增加14倍，k（cat）减少12倍，表明该酶保守的亲水性C末端可能在底物结合和催化中起作用。
Prenyltransferases that catalyze the fundamental chain elongation reaction in the isoprenoid biosynthetic pathway contain several highly conserved amino acids, including two aspartate-rich regions thought to be involved in substrate binding and catalysis. We report a study of site-directed mutants for yeast farnesyl-diphosphate synthase (FPPSase; geranyl-diphosphate:isopentenyl-diphosphate, EC 2.5.1.10), a prenyltransferase that catalyzes the sequential 1'-4 coupling of isopentenyl diphosphate (IPP) with dimethylallyl diphosphate and geranyl diphosphate. A recombinant form of FPPSase extended by a C-terminal -Glu-Glu-Phe alpha-tubulin epitope (EEF in single-letter amino acid code) was engineered to facilitate rapid purification of the enzyme by immunoaffinity chromatography and to remove traces of contaminating activity from wild-type FPPSase in the Escherichia coli host. Ten site-directed mutants were constructed in FPPSase::EEF. The six aspartates in domain I (at positions 100, 101, and 104) and domain II (at positions 240, 241, and 244) were changed to alanine (mutants designated D100A, D101A, D104A, D240A, D241A, and D244A); three arginine residues.were changed, Arg-109 and Arg-110 to glutamine and Arg-350 to alanine (mutants designated R109Q, R110Q, and R350A); and Lys-254 was converted to alanine (mutant designated K254A). Mutations of the aspartatic residues and nearby arginine residues in domain I and Asp-240 and Asp-241 in domain II drastically lowered the catalytic activity of FPPSase::EEF. The D244A and K254A mutants were substantially less active, while k(cat) and the Michaelis constants for the R350A mutant were similar to those of FPPSase::EEF. Addition of an -EEF epitope to the C terminus of wild-type FPPSase resulted in a 14-fold increase of K(m)IPP and a 12-fold decrease of k(cat), suggesting that the conserved hydrophilic C terminus of the enzyme may have a role in substrate binding and catalysis.