Structural‐based mutational analysis of d ‐aminoacylase from Alcaligenes faecalis DA1

Abstract

d ‐Aminoacylase is an attractive candidate for commercial production of d ‐amino acids through its catalysis in the zinc‐assistant hydrolysis of N ‐acyl‐ d ‐amino acids. We report here the cloning, expression, and structural‐based mutation of the d ‐aminoacylase from Alcaligenes faecalis DA1. A 1,007‐bp PCR product amplified with degenerate primers, was used to isolate a 4‐kb genomic fragment, encoding a 484‐residue d ‐aminoacylase. The enzyme amino‐terminal segment shared significant homology within a variety of enzymes including urease. The structural fold was predicted by 3D‐PSSM to be similar to urease and dihydroorotase, which have grouped into a novel α/β‐barrel amidohydrolase superfamily with a virtually indistinguishable binuclear metal centers containing six ligands, four histidines, one aspartate, and one carboxylated lysine. Three histidines, His‐67, His‐69, and His‐250, putative metal ligands in d ‐aminoacylase, have been mutated previously, the remaining histidine (His‐220) and aspartate (Asp‐366) Asp‐65, and four cysteines were then characterized. Substitution of Asp‐65, Cys‐96, His‐220, and Asp‐366 with alanine abolished the enzyme activity. The H220A mutant bound approximately half the normal complement of zinc ion as did H250N. However, the C96A mutant showed little zinc‐binding ability, revealing that Cys‐96 may replace the carboxylated lysine to serve as a bridging ligand. According to the urease structure, the conserved amino‐terminal segment including Asp‐65 may be responsible for structural stabilization.