Mutational and structural analysis of L-N-carbamoylase reveals 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 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.
Departamento de Química Física, Bioquímica y Química Inorgánica, Universidad de Almería, Almería, Spain.
Sergio
S
Martínez-Rodríguez
Abel
A
García-Pino
Francisco Javier
FJ
Las Heras-Vázquez
Josefa María
JM
Clemente-Jiménez
Felipe
F
Rodríguez-Vico
Juan M
JM
García-Ruiz
Remy
R
Loris
Jose Antonio
JA
Gavira
D000581:Amidohydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019943:Amino Acid Substitution; D020134:Catalytic Domain; D017124:Conserved Sequence; D018360:Crystallography, X-Ray; D001411:Geobacillus stearothermophilus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D055503:Protein Multimerization; D013379:Substrate Specificity
D050505:Mutant Proteins; D000581:Amidohydrolases; C104561:N-carbamoyl-L-amino-acid hydrolase