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

AuthorsMartínez Rodríguez, Sergio; García-Pino, Abel; Las Heras-Vázquez, F. J.; Clemente-Jiménez, J. M.; Rodríguez-Vico, F.; García Ruiz, Juan Manuel ; Loris, Remy; Gavira Gallardo, J. A.
Issue Date2012
PublisherAmerican Society for Microbiology
CitationJournal of Bacteriology 194: 5759-5768 (2012)
AbstractN-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. © 2012, American Society for Microbiology.
Publisher version (URL)http://dx.doi.org/10.1128/JB.01056-12
Identifiersdoi: 10.1128/JB.01056-12
issn: 0021-9193
Appears in Collections:(IACT) Artículos
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