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Protein Science (2006), 15:2729-2738. Published by Cold Spring Harbor Laboratory Press. Copyright © 2006 The Protein Society
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Site-directed mutagenesis indicates an important role of cysteines 76 and 181 in the catalysis of hydantoin racemase from Sinorhizobium meliloti

Sergio Martínez-Rodríguez1,4, Montserrat Andújar-Sánchez1, Jose L. Neira2,3, Josefa M. Clemente-Jiménez1, Vicente Jara-Pérez1, Felipe Rodríguez-Vico1, and Francisco J. Las Heras-Vázquez1

1 Departamento Química Física, Bioquímica y Química Inorgánica, Universidad de Almería, 04120 Almería, Spain
2 Instituto de Biología Molecular y Celular, Universidad Miguel Hernández, 03202 Elche (Alicante), Spain
3 Instituto de Biocomputación y Física de los Sistemas Complejos, 50009 Zaragoza, Spain

(RECEIVED July 20, 2006; FINAL REVISION September 20, 2006; ACCEPTED September 25, 2006)

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.

Keywords: mutagenesis; hydantoin racemase; reaction mechanism; isothermal titration calorimetry; fluorescence; circular dichroism


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