Evaluation of substrate promiscuity of an L-carbamoyl amino acid amidohydrolase from Geobacillus stearothermophilus CECT43.
N-carbamoyl-amino-acid amidohydrolase (also known as N-carbamoylase) is the stereospecific enzyme responsible for the chirality of the D- or L-amino acid obtained in the "Hydantoinase Process." This process is based on the dynamic kinetic resolution of D,L-5-monosubstituted hydantoins. In this work, we have demonstrated the capability of a recombinant L-N-carbamoylase from the thermophilic bacterium Geobacillus stearothermophilus CECT43 (BsLcar) to hydrolyze N-acetyl and N-formyl-L-amino acids as well as the known N-carbamoyl-L-amino acids, thus proving its substrate promiscuity. BsLcar showed faster hydrolysis for N-formyl-L-amino acids than for N-carbamoyl and N-acetyl-L-derivatives, with a catalytic efficiency (k(cat)/K(m)) of 8.58 x 10(5), 1.83 x 10(4), and 1.78 x 10(3) (s(-1) M(-1)), respectively, for the three precursors of L-methionine. Optimum reaction conditions for BsLcar, using the three N-substituted-L-methionine substrates, were 65 degrees C and pH 7.5. In all three cases, the metal ions Co(2+), Mn(2+), and Ni(2+) greatly enhanced BsLcar activity, whereas metal-chelating agents inhibited it, showing that BsLcar is a metalloenzyme. The Co(2+)-dependent activity profile of the enzyme showed no detectable inhibition at high metal ion concentrations.
Pozo-Dengra
Joaquín
J
Dept. de Química-Física, Bioquímica y Química Inorgánica. Edificio C.I.T.E. I., Universidad de Almería, La Cañada de San Urbano, Almería, Spain.
Martínez-Gómez
Ana Isabel
AI
Martínez-Rodríguez
Sergio
S
Clemente-Jiménez
Josefa María
JM
Rodríguez-Vico
Felipe
F
Las Heras-Vázquez
Francisco Javier
FJ
D000581:Amidohydrolases / Q000235:genetics / Q000378:metabolism*; D001411:Geobacillus stearothermophilus / Q000201:enzymology* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D013379:Substrate Specificity; D013696:Temperature
D000581:Amidohydrolases; C451299:N-carbamoylamino acid amidohydrolase