Metal-triggered changes in the stability and secondary structure of a tetrameric dihydropyrimidinase: A biophysical characterization |
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Authors: | Sergio Martínez-Rodríguez José A Encinar Estefanía Hurtado-Gómez Jesús Prieto Josefa M Clemente-Jiménez Francisco J Las Heras-Vázquez Felipe Rodríguez-Vico José L Neira |
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Institution: | 1. Laboratorium voor Ultrastructuur, Vrije Universiteit Brussels, Pleinlaan 2, B-1050 Brussels, Belgium;2. Department of Molecular and Cellular Interactions, VIB, Pleinlaan 2, B-1050 Brussels, Belgium;3. Instituto de Biología Molecular y Celular, Universidad Miguel Hernández, 03202 Elche, Alicante, Spain;4. Structural Biology and Biocomputing Programme, Centro Nacional de Investigaciones Oncológicas (CNIO), 28007 Madrid, Spain;5. Departamento de Química Física, Bioquímica y Química Inorgánica, Universidad de Almería, 04120 Almería, Spain;6. Biocomputation and Complex Systems Physics Institute, 50009 Zaragoza, Spain |
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Abstract: | Dihydropyrimidinase is involved in the reductive pathway of pyrimidine degradation, catalysing the reversible hydrolysis of the cyclic amide bond (–CO–NH–) of 5,6-dihydrouracil and 5,6-dihydrothymine to the corresponding N-carbamoyl-β-amino acids. This enzyme is an attractive candidate for commercial production of D-amino acids, which are used in the production of semi-synthetic β-lactams, antiviral agents, artificial sweeteners, peptide hormones and pesticides. We have obtained the crystal structure of the dihydropyrimidinase from Sinorhizobium meliloti (SmelDhp) in the presence of zinc ions, but we have not been able to obtain good diffracting crystals in its absence. Then, the role of the ion in the structure of the protein, and in its stability, remains to be elucidated. In this work, the stability and the structure of SmelDhp have been studied in the absence and in the presence of zinc. In its absence, the protein acquired a tetrameric functional structure at pH ∼ 6.0, which is stable up to pH ∼ 9.0, as concluded from fluorescence and CD. Chemical-denaturation occurred via a monomeric intermediate with non-native structure. The addition of zinc caused: (i) an increase of the helical structure, and changes in the environment of aromatic residues; and, (ii) a higher thermal stability. However, chemical-denaturation still occurred through a monomeric intermediate. This is the first hydantoinase whose changes in the stability and in the secondary structure upon addition of zinc are described and explained, and one of the few examples where the zinc exclusively alters the secondary helical structure and the environment of some aromatic residues in the protein, leaving unchanged the quaternary structure. |
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Keywords: | ANS 8-anilinonaphtalene-1-sulfonic acid ASA accessible solvent area DSC differential scanning calorimetry GdmCl guanidine hydrochloride [GdmCl]1/2 the denaturation midpoint of the chemical-denaturation FTIR Fourier transform infrared spectroscopy Tm the thermal and calorimetric denaturation midpoints SmelDhp dihydropyrimidinase from Sinorhizobium meliloti Ve elution volume |
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