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

TitleMutational and structural analysis of L-N-carbamoylase reveals new insights into a peptidase M20/M25/M40 family member.
Publication TypeJournal Article
Year of Publication2012
AuthorsMartinez-Rodriguez, S., A. Garcia-Pino, F. Javier Las Heras-Vázquez, J. María Clemente-Jiménez, F. Rodríguez-Vico, J. M. García-Ruiz, R. Loris, and J. Antonio Gavira
JournalJ Bacteriol
Volume194
Issue21
Pagination5759-68
Date Published2012 Nov
ISSN1098-5530
KeywordsAmidohydrolases, Amino Acid Substitution, Catalytic Domain, Conserved Sequence, Crystallography, X-Ray, Geobacillus stearothermophilus, Models, Molecular, Mutagenesis, Site-Directed, Mutant Proteins, Protein Binding, Protein Conformation, Protein Multimerization, Substrate Specificity
Abstract

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 a resolution of 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 revealed their relevance to substrate binding. We also found an unexpectedly conserved arginine residue (Arg(234) in BsLcar), proven to be critical for dimerization of the enzyme. The mutation of this sole residue resulted in a total loss of activity and prevented the formation of the dimer in BsLcar. Comparative studies revealed that the dimerization domain of the peptidase M20/M25/M40 family is a "small-molecule binding domain," allowing further evolutionary considerations for this enzyme family.

DOI10.1128/JB.01056-12
Alternate JournalJ. Bacteriol.
PubMed ID22904279
PubMed Central IDPMC3486127