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

Abstract

Background: N-carbamyl- d-amino acid amidohydrolase (DCase) catalyzes the hydrolysis of N-carbamyl- d-amino acids to the corresponding d-amino acids, which are useful intermediates in the preparation of β-lactam antibiotics. To understand the catalytic mechanism of N-carbamyl- d-amino acid hydrolysis, the substrate specificity and thermostability of the enzyme, we have determined the structure of DCase from Agrobacterium sp. strain KNK712. Results: The crystal structure of DCase has been determined to 1.7 Å resolution. The enzyme forms a homotetramer and each monomer consists of a variant of the α+β fold. The topology of the enzyme comprises a sandwich of parallel β sheets surrounded by two layers of α helices, this topology has not been observed in other amidohydrolases such as the N-terminal 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 activation by Glu46. DCase shows only weak sequence similarity with a family of amidohydrolases, including β-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 for this family of enzymes.