title: Molecular cloning and biochemical characterization of L-N-carbamoylase from Sinorhizobium meliloti CECT4114.
abstract: An N-carbamoyl-L-amino acid amidohydrolase (L-N-carbamoylase) from Sinorhizobium meliloti CECT 4114 was cloned and expressed in Escherichia coli. The recombinant enzyme catalyzed the hydrolysis of N-carbamoyl alpha-amino acid to the corresponding free amino acid, and its purification has shown it to be strictly L-specific. The enzyme showed broad substrate specificity, and it is the first L-N-carbamoylase that hydrolyses N-carbamoyl-L-tryptophan as well as N-carbamoyl L-amino acids with aliphatic substituents. The apparent Km values for N-carbamoyl-L-methionine and tryptophan were very similar (0.65 +/- 0.09 and 0.69 +/- 0.08 mM, respectively), although the rate constant was clearly higher for the L-methionine precursor (14.46 +/- 0.30 s(-1)) than the L-tryptophan one (0.15 +/- 0.01 s(-1)). The enzyme also hydrolyzed N-formyl-L-methionine (kcat/Km = 7.10 +/- 2.52 s(-1) x mM(-1)) and N-acetyl-L-methionine (kcat/Km = 12.16 +/- 1.93 s(-1) x mM(-1)), but the rate of hydrolysis was lower than for N-carbamoyl-L-methionine (kcat/Km = 21.09 +/- 2.85). This is the first L-N-carbamoylase involved in the 'hydantoinase process' that has hydrolyzed N-carbamoyl-L-cysteine, though less efficiently than N-carbamoyl-L-methionine. The enzyme did not hydrolyze ureidosuccinic acid or 3-ureidopropionic acid. The native form of the enzyme was a homodimer with a molecular mass of 90 kDa. The optimum conditions for the enzyme were 60 degrees C and pH 8.0. Enzyme activity required the presence of divalent metal ions such as Ni2+, Mn2+, Co2+ and Fe2+, and five amino acids putatively involved in the metal binding were found in the amino acid sequence.
authors.affiliation: Departamento de Química-Física, Bioquímica y Química Inorgánica, I. Universidad de Almería, La Cañada de San Urbano, Almería, España.
authors.forename: Sergio
authors.initials: S
authors.lastname: Martínez-Rodríguez
authors.forename: Josefa María
authors.initials: JM
authors.lastname: Clemente-Jiménez
authors.forename: Felipe
authors.initials: F
authors.lastname: Rodríguez-Vico
authors.forename: Francisco Javier
authors.initials: FJ
authors.lastname: Las Heras-Vázquez
mesh_terms: D000581:Amidohydrolases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D002219:Carbamates / Q000378:metabolism; D003001:Cloning, Molecular; D003067:Coenzymes / Q000378:metabolism; D019281:Dimerization; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D008670:Metals / Q000494:pharmacology; D008715:Methionine / Q000031:analogs & derivatives / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009239:N-Formylmethionine / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D016962:Sinorhizobium meliloti / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity; D013696:Temperature; D014364:Tryptophan / Q000031:analogs & derivatives / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D002219:Carbamates; D003067:Coenzymes; D008670:Metals; D011994:Recombinant Proteins; C072457:N-carbamylmethionine; D001224:Aspartic Acid; D009239:N-Formylmethionine; C052867:N-carbamyltryptophan; D014364:Tryptophan; D008715:Methionine; D000581:Amidohydrolases; C451299:N-carbamoylamino acid amidohydrolase; C020047:dihydropyrimidinase; C006893:ureidosuccinic acid