Crystal Structure 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 hydrolysis of N-acyl-D-amino acids. We report here the first D-aminoacylase crystal structure from A. faecalis at 1.5-A resolution. The protein comprises a small [beta]-barrel, and a catalytic ([beta][alpha])8-barrel with a 63-residue insertion. The enzyme structure shares significant similarity to the [alpha]/[beta]-barrel amidohydrolase superfamily, in which the [beta]-strands in both barrels superimpose well. Unexpectedly, the enzyme binds two zinc ions with widely different affinities, although only the tightly bound zinc ion is required for activity. One zinc ion is coordinated by Cys96, His220, and His250, while the other is loosely chelated by His67, His69, and Cys96. This is the first example of the metal ion coordination by a cysteine residue in the superfamily. Therefore, D-aminoacylase defines a novel subset and is a mononuclear zinc metalloenzyme but containing a binuclear active site. The preferred substrate was modeled into a hydrophobic pocket, revealing the substrate specificity and enzyme catalysis. The 63-residue insertion containing substrate-interacting residues may act as a gate controlling access to the active site, revealing that the substrate binding would induce a closed conformation to sequester the catalysis from solvent.