Structural-based mutational analysis of D-aminoacylase from Alcaligenes faecalis DA1.
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 alpha/beta-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.
Hsu
Cheng-Sheng
CS
Institute of Biochemistry, National Yang-Ming University, Taipei, Taiwan.
Lai
Wen-Lin
WL
Chang
Wei-Wei
WW
Liaw
Shwu-Huey
SH
Tsai
Ying-Chieh
YC
D000421:Alcaligenes / Q000201:enzymology* / Q000235:genetics; D000581:Amidohydrolases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D001665:Binding Sites; D003001:Cloning, Molecular; D003545:Cysteine / Q000737:chemistry; D004252:DNA Mutational Analysis; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D016415:Sequence Alignment
D001426:Bacterial Proteins; D008024:Ligands; D000581:Amidohydrolases; C019672:aminoacylase I; D003545:Cysteine