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Structural-based mutational analysis of D-aminoacylase from Alcaligenes faecalis DA1

¹ Institute of Biochemistry
² Department of Life Science, National Yang-Ming University, Taipei, Taiwan
³ Department of Biochemistry, National Taipei College of Nursing, Taipei, Taiwan
⁴ Department of Medical Research and Education, Taipei Veterans General Hospital, Taipei, Taiwan

Reprint requests to: Shwu-Huey Liaw or Ying-Chieh Tsai, Institute of Biochemistry, National Yang-Ming University, 155, Sec. 2, Li-Nong Street, Pei-Tou, Taipei 11221, Taiwan; e-mail: shliaw{at}ym.edu.tw or tsaiyc{at}ym.edu.tw; fax: 886–2–2820–2449.

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 /ß-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.

Keywords: d-Aminoacylase; /ß-barrel amidohydrolase superfamily; zinc ligation

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