Structural and functional analysis of microbial enzymes catalyzing defluorination and fluorination

研究了氟乙酸脱卤酶和L-2-卤酸脱卤酶的结构和功能。两种酶反应都分两步进行。在第一步中，酶的活性位点天冬氨酸残基的羧酸酯基团攻击底物的α-碳原子以从底物释放卤离子，导致形成由酶和底物组成的酯中间体。在第二步中，酯中间体水解以恢复活性位点羧酸酯基团并产生羟基链烷酸。我们确定的晶体结构的酶-底物复合物的L-2-卤酸脱卤酶，以及它的酯中间体使用突变酶，不能有效地催化第二步反应。特别是，我们确定了残基识别的羧酸基团的基板，并接受从基板释放的卤素离子。对于氟乙酸脱卤酶，我们用羟胺和氨水进行了副催化失活实验。我们发现Aspl 05被这些亲核试剂修饰，表明该残基是催化残基。我们预测的三维结构的氟乙酸脱卤酶的同源建模，并发现His 272，这是建议激活一个水分子的酯中间体的水解，位于附近的Asp 105。Argl 06和Trp 151被认为接受从基底释放的氟离子。活性部位主要由疏水性和碱性氨基酸残基组成。这种环境可能有助于Aspl 05的羧酸酯基团的高亲核性，并且能够裂解碳-氟键。而L-2-卤酸脱卤酶的活性中心主要由亲水性氨基酸残基组成，不能催化氟乙酸的水解。

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.