Structural and functional analysis of microbial enzymes catalyzing defluorination and fluorination

催化脱氟和氟化的微生物酶的结构和功能分析

基本信息

批准号：
09460049
负责人：
ESAKI Nobuyoshi
金额：
$ 4.8万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

项目摘要

Structures and functions of fluoroacetate dehalogenase and L-2-haloacid dehalogenase were studied. Both enzyme reactions proceed in two steps. In the first step, a carboxylate group of the active-site aspartate residue of the enzyme attacks the alpha-carbon atom of the substrate to release a halide ion from the substrate, leading to the formation of an ester intermediate consisting of the enzyme and the substrate. In the second step, the ester intermediate is hydrolyzed to restore the active-site carboxylate group and produce hydroxyalkanoic acid. We determined the crystal structure of an enzyme-substrate complex of L-2-haloacid dehalogenase as well as its ester intermediate using a mutant enzyme that does not catalyze the second step reaction efficiently. In particular, we identified the residues that recognize the carboxylate group of the substrate and accept the halide ion released from the substrate. As to fluoroacetate dehalogenase, we performed a paracatalytic inactivation experiment using hydroxylamine and ammonia. We found that Aspl05 was modified by these nucleophiles, indicating that this residue is a catalytic residue. We Predicted the three dimensional structure of fluoroacetate dehalogenase by homology modeling, and found that His272, which is proposed to activate a water molecule for hydrolysis of the ester intermediate, is located in the vicinity of Asp 105. Argl06 and Trp151 were suggested to accept fluoride ion released from the substrate. Active site is mainly composed of hydrophobic and basic amino acid residues. This environment probably contributes to the high nucleophilicity of the carboxylate group of Aspl05, and enables the cleavage of the carbon-fluoride bond. In contrast, the active site of L-2-haloacid dehalogenase, which cannot catalyze the hydrolysis of fluoroacetate, is mainly composed of hydrophilic amino acid residues.

研究了氟乙酸脱核酶和L-2-卤素脱核酶的结构和功能。这两种酶反应都以两个步骤进行。在第一步中，酶的活性位点天冬氨酸残基的羧酸盐攻击底物的α-碳原子，从底物释放卤离子离子，从而形成由酶和底物组成的酯中间体。在第二步中，将酯中间体水解为恢复活性位点羧酸酯基并产生羟基烷酸。我们使用不会有效地催化第二步反应的突变酶确定了L-2-半酸脱核酶的酶 - 基底络合物的晶体结构及其酯中间体。特别是，我们确定了识别底物的羧酸酯基的残基，并接受从底物释放的卤化物离子。至于氟乙酸脱核酶，我们使用羟胺和氨进行了屈催化的灭活实验。我们发现ASPL05通过这些亲核试剂进行了修饰，表明该残基是催化残基。我们通过同源性建模预测了氟乙酸脱核酶的三维结构，并发现HIS272被提议激活水分子以水解酯中间体的水解，位于ASP105。ASP105。ARGL06和TRP151的附近，建议接受氟化物从二元中释放出来。活性位点主要由疏水和碱性氨基酸残基组成。这种环境可能有助于ASPL05的羧酸盐基团的高亲核性，并实现碳氟化物键的裂解。相比之下，无法催化氟乙酸水解的L-2-半酸脱核酶的活性位点主要由亲水性氨基酸残基组成。