Crystal structure of N-carbamyl-D-amino acid amidohydrolase with a novel catalytic framework common to amidohydrolases

Background: N-carbamyl-D-amino acid amidohydrolase (DCase) catalyzes the hy drolysis of N-carbamyl-D-amino acids to the corresponding D-amino acids, wh ich are useful intermediates in the preparation of p-lactam antibiotics, To understand the catalytic mechanism of N-carbamyl-D-amino acid hydrolysis, the substrate specificity and thermostability of the enzyme, we have determ ined the structure of DCase from Agrobacterium sp. strain KNK712. Results: The crystal structure of DCase has been determined to 1.7 Angstrom resolution. The enzyme forms a homotetramer and each monomer consists of a variant of the alpha+beta fold. The topology of the enzyme comprises a san dwich of parallel beta sheets surrounded by two layers of alpha helices, th is topology has not been observed in other amidohydrolases such as the N-te rminal nucleophile (Ntn) hydrolases. Conclusions: The catalytic center could be identified and consists of Glu46 , Lys126 and Cys171. Cys171 was found to be the catalytic nucleophile, and its nucleophilic character appeared to be increased through general-base ac tivation by Glu46. DGase shows only weak sequence similarity with a family of amidohydrolases, including beta-alanine synthase, aliphatic amidases and nitrilases, but might share highly conserved residues in a novel framework , which could provide a possible explanation for the catalytic mechanism fo r this family of enzymes.