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Publication Abstract from PubMed

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. In 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 1.49 A resolution. 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 electron 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. Although it was difficult to locate 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. This article is protected by copyright. All rights reserved.

Crystal structure of D-stereospecific amidohydrolase from Streptomyces sp. 82F2: insight into the structural factors for substrate specificity.,Arima J, Shimone K, Miyatani K, Tsunehara Y, Isoda Y, Hino T, Nagano S FEBS J. 2015 Oct 29. doi: 10.1111/febs.13579. PMID:26513520^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.