Mutational and Structural Analysis of L-N-Carbamoylase Reveals New Insights into a Peptidase M20/M25/M40 Family Member
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Martínez Rodríguez, Sergio; Garcia-Pino A.; Las Heras Vázquez, Francisco Javier; Clemente Jiménez, Josefa María; Rodríguez Vico, Felipe; [et al.]Fecha
2012Resumen
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 (Arg234 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 furthe...
Palabra/s clave
L-N-Carbamoylase
Peptidase