A conserved histidine is essential for glycerolipid acyltransferase catalysis
A conserved histidine is essential for glycerolipid acyltransferase catalysis
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DOI：
10.1128/jb.180.6.1425-1430.1998
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发表时间：
1998-03-01
影响因子：
3.2
通讯作者：
Rock, CO
中科院分区：
文献类型：
--
作者：
Heath, RJ;Rock, CO
对膜结合甘油脂酰基转移酶的序列分析表明，来自细菌、植物和动物的蛋白质共享一个高度保守的结构域，其中包含不变组氨酸和天冬氨酸残基，在HX4D构型中由四个较不保守的残基分开。我们通过位点定向诱变研究了不变组氨酸残基在酰基转移酶催化中的作用。大肠杆菌的sn-甘油-3-磷酸酰基转移酶（PlsB）和双功能2-酰基-甘油磷酸乙醇胺酰基转移酶/酰基-酰基载体蛋白合成酶（Aas）， PlsB[H306A]和Aas[H36A]突变体均缺乏酰基转移酶活性。然而，Aas[H36A]突变体保留了显著的酰基酰基载体蛋白合成酶活性，说明酰基转移酶活性的缺乏与H36A取代特异性相关，HX4D模式中不变的天冬氨酸残基也很重要，PlsB中用谷氨酸取代天冬氨酸311导致酶的催化活性显著降低。在这个位置替换丙氨酸消除了酰基转移酶的活性；然而，PlsB[D311A]突变蛋白没有组装到膜上，这表明天冬氨酸311对于酰基转移酶的正确折叠和膜插入也很重要。这些数据与甘油脂酰基转移酶催化的机制一致，其中不变组氨酸作为酰基受体羟基部分去质子化的一般碱。
Sequence analysis of membrane-bound glycerolipid acyltransferases revealed that proteins from the bacterial, plant, and animal kingdoms share a highly conserved domain containing invariant histidine and aspartic acid residues separated by four less conserved residues in an HX4D configuration, We investigated the role of the invariant histidine residue in acyltransferase catalysis by site-directed mutagenesis of two representative members of this family, the sn-glycerol-3-phosphate acyltransferase (PlsB) and the bifunctional 2-acyl-glycerophosphoethanolamine acyltransferase/acyl-acyl carrier protein synthetase (Aas) of Escherichia coli, Both the PlsB[H306A] and Aas[H36A] mutants lacked acyltransferase activity. However, the Aas[H36A] mutant retained significant acyl-acyl carrier protein synthetase activity, illustrating that the lack of acyltransferase activity was specifically associated with the H36A substitution, The invariant aspartic acid residue in the HX4D pattern was also important, The substitution of aspartic acid 311 with glutamic acid in PlsB resulted in an enzyme with significantly reduced catalytic activity. Substitution of an alanine at this position eliminated acyltransferase activity; however, the PlsB[D311A] mutant protein did not assemble into the membrane, indicating that aspartic acid 311 is also important for the proper folding and membrane insertion of the acyltransferases. These data are consistent with a mechanism for glycerolipid acyltransferase catalysis where the invariant histidine functions as a general base to deprotonate the hydroxyl moiety of the acyl acceptor.