Biochemical characterization of a novel hydantoin racemase from Agrobacterium tumefaciens C58.
A novel hydantoin racemase gene of Agrobacterium tumefaciens C58 (AthyuA2) has been cloned and expressed in Escherichia coli BL21. The recombinant protein was purified in a one-step procedure and showed an apparent molecular mass of 27000 Da in SDS-gel electrophoresis. Size exclusion chromatography analysis determined a molecular mass of approximately 100000 Da, suggesting that the native enzyme is a tetramer. The optimum pH and temperature for hydantoin racemase activity were 7.5 and 55 degrees C, respectively, with L-5-ethylhydantoin as substrate. Enzyme activity was strongly inhibited by Cu(2+) and Hg(2+). No effect on enzyme activity was detected with any other divalent cations, EDTA or DTT, suggesting that it is not a metalloenzyme. Kinetic studies showed the preference of the enzyme for hydantoins with short rather than long aliphatic side chains or hydantoins with aromatic rings.
Martínez-Rodríguez
Sergio
S
Departamento de Química-Física, Bioquímica y Química Inorgánica. Edificio CITEI Universidad de Almería, La Cañada de San Urbano, 04120 Almeria, Spain.
Las Heras-Vázquez
Francisco Javier
FJ
Clemente-Jiménez
Josefa María
JM
Rodríguez-Vico
Felipe
F
D016960:Agrobacterium tumefaciens / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D004229:Dithiothreitol / Q000737:chemistry; D004492:Edetic Acid / Q000737:chemistry; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008670:Metals / Q000737:chemistry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D019998:Racemases and Epimerases / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D017422:Sequence Analysis, DNA; D013237:Stereoisomerism; D013696:Temperature; D013997:Time Factors
D008670:Metals; D011994:Recombinant Proteins; D004492:Edetic Acid; D019998:Racemases and Epimerases; C074773:hydantoin racemase; D004229:Dithiothreitol