Structural and biochemical characterisation of the N-Carbamoyl-beta-Alanine Amidohydrolases from Rhizobium radiobacter MDC 8606

Abstract

N-Carbamoyl-{beta}-Alanine Amidohydrolase (C{beta}AA) constitute one of the most important groups of industrially relevant enzymes used in production of optically pure amino acids and derivatives. In this study, a N-carbamoyl-{beta}-alanine amidohydrolase encoding gene from Rhizobium radiobacter MDC 8606 was cloned and overexpressed in Escherichia coli. The purified recombinant enzyme (RrC{beta}AA) showed a specific activity of 14 U/mg using N-carbamoyl-{beta}-alanine as a substrate with an optimum activity of 55{degrees}C at pH 8.0. In this work, we report also the first prokaryotic N-carbamoyl-{beta}-alanine amidohydrolases structure at a resolution of 2.0 [A]. A discontinuous catalytic domain and a dimerization domain attached through a flexible hinge region at the domain interface has been revealed. We have found that the ligand is interacting with a conserved glutamic acid (Glu131), histidine (H385) and arginine (Arg291) residues. Studies let us to explain the preference on the enzyme for linear carbamoyl substrates as large carbamoyl substrates cannot fit in the active site of the enzyme. This work envisages the use of RrC{beta}AA from the Rhizobium radiobacter MDC 8606 for the industrial production of L--, L-{beta}-, and L-{gamma} - amino acids. The structural analysis provides new insights on enzyme-substrate interaction, which shed light on engineering of N-carbamoyl-{beta}-alanine amidohydrolases for high catalytic activity and broad substrate specificity.