Site-directed mutagenesis indicates an important role of cysteines 76 and 181 in the catalysis of hydantoin racemase from Sinorhizobium meliloti.
Hydantoin racemase enzyme plays a crucial role in the reaction cascade known as "hydantoinase process." In conjunction with a stereoselective hydantoinase and a stereospecific carbamoylase, it allows the total conversion from D,L-5-monosubstituted hydantoins, with a low rate of racemization, to optically pure D- or L-amino acids. Residues Cys76 and Cys181 belonging to hydantoin racemase from Sinorhizobium meliloti (SmeHyuA) have been proved to be involved in catalysis. Here, we report biophysical data of SmeHyuA Cys76 and Cys181 to alanine mutants, which point toward a two-base mechanism for the racemization of 5-monosubstituted hydantoins. The secondary and the tertiary structure of the mutants were not significantly affected, as shown by circular dichroism. Calorimetric and fluorescence experiments have shown that Cys76 is responsible for recognition and proton retrieval of D-isomers, while Cys181 is responsible for L-isomer recognition and racemization. This recognition process is further supported by measurements of protein stability followed by chemical denaturation in the presence of the corresponding compound.
Martínez-Rodríguez
Sergio
S
Departamento Química Física, Bioquímica y Química Inorgánica, Universidad de Almería, 04120 Almería, Spain.
Andújar-Sánchez
Montserrat
M
Neira
Jose L
JL
Clemente-Jiménez
Josefa M
JM
Jara-Pérez
Vicente
V
Rodríguez-Vico
Felipe
F
Las Heras-Vázquez
Francisco J
FJ
D000595:Amino Acid Sequence; D001665:Binding Sites; D002151:Calorimetry / Q000379:methods; D002384:Catalysis; D002942:Circular Dichroism / Q000379:methods; D003001:Cloning, Molecular; D003198:Computer Simulation; D017124:Conserved Sequence; D003545:Cysteine / Q000502:physiology*; D005453:Fluorescence; D019791:Guanidine / Q000494:pharmacology; D008954:Models, Biological; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D050505:Mutant Proteins / Q000737:chemistry / Q000187:drug effects; D011485:Protein Binding; D011487:Protein Conformation / Q000187:drug effects; D017510:Protein Folding; D019998:Racemases and Epimerases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid; D016962:Sinorhizobium meliloti / Q000737:chemistry / Q000201:enzymology*
D050505:Mutant Proteins; D019998:Racemases and Epimerases; C074773:hydantoin racemase; D019791:Guanidine; D003545:Cysteine