Mutational and structural analysis of L-N-carbamoylase: new insights into a peptidase M20/M25/M40 family member

N-Carbamoyl-L-amino acid amidohydrolases (L-carbamoylases) are important industrial enzymes used in kinetic resolution of racemic mixtures of N-carbamoyl-amino acids due to their strict enantiospecificity. In this work, we report the first L-carbamoylase structure belonging to Geobacillus stearothermophilus CECT43 (BsLcar) at 2.7 Å. Structural analysis of BsLcar and several members of the peptidase M20/M25/M40 family confirmed the expected conserved residues at the active site in this family and site-directed mutagenesis reveal their relevance in substrate binding. We also found an unexpectedly conserved arginine residue (Arg234 in BsLcar), proven to be critical for dimerization of the enzyme. The mutation of this sole residue resulted in total loss of activity, and avoided the formation of the dimer in BsLcar. Comparative studies revealed the dimerization domain of the peptidase M20/M25/M40 family as a "small molecule binding domain", allowing further evolutionary considerations for this enzyme family.