Structural and functional analysis of microbial enzymes catalyzing defluorination and fluorination

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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.