D-Stereospecific amidohydrolase (DAH) from Streptomyces sp. 82F2, which catalyzes amide bond formation from D-aminoacyl esters and L-amino acids (aminolysis), can be used to synthesize short peptides with a DL-configuration. We found that DAH can use 1,8-diaminooctane and other amino compounds as acyl acceptors in the aminolysis reaction. Low concentrations of 1,8-diaminooctane inhibited acyl-DAH intermediate formation. By contrast, excess 1,8-diaminooctane promoted aminolysis by DAH, producing D-Phe-1,8-diaminooctane via nucleophilic attack of the diamine on enzyme-bound D-Phe. To clarify the mechanism of substrate specificity and amide bond formation by DAH, the crystal structure of the enzyme that binds 1,8-diaminooctane was determined at a resolution of 1.49A. Comparison of the DAH crystal structure with those of other members of the S12 peptidase family indicated that the substrate specificity of DAH arises from its active site structure. The 1,8-diaminooctane molecule binds at the entrance of the active site pocket. The electrkon density map showed that another potential 1,8-diaminooctane binding site, probably with lower affinity, is present close to the active site. The enzyme kinetics and structural comparisons suggest that the location of enzyme-bound diamine can explain the inhibition of the acyl-enzyme intermediate formation, although the bound diamine is too far from the active site for aminolysis. Despite difficulty in locating the diamine binding site for aminolysis definitively, we propose that the excess diamine also binds at or near the second binding site to attack the acyl-enzyme intermediate during aminolysis.

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D-Stereospecific amidohydrolase (DAH) from Streptomyces sp. 82F2 has potential utility for the synthesis of D/L configuration dipeptides by an aminolysis reaction. Structural comparison of DAH with substrate-bound D-amino acid amidase revealed that three residues located in the active site pocket of DAH (Thr145, Ala267, and Gly271) might be involved in interactions with D-phenylalanine substrate. We substituted Ala267 and Gly271, which are located at the bottom of the hydrophobic pocket of DAH, with Phe and observed changes in the stereoselectivity and specific activity toward the free and acetylated forms of D/L-Phe-methyl esters. In contrast, the mutation of Thr145, which likely supplies negative charge for recognition of the amino group of the substrate, hardly affected the stereoselectivity of the enzyme. A similar effect was observed in an investigation of hydrolysis and aminolysis reactions using the acetylated forms of D/L-Phe-methyl esters and 1,8-diaminooctane as an acyl-donor and acyl-acceptor, respectively. Substrate binding by DAH was disrupted by the mutation of Ala267 to Val or Trp and kinetic analysis showed that the hydrophobicity of the bottom of the active site pocket (Ala267 and Gly271) is important for both stereoselectivity and recognition of hydrophobic substrates.