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Metal-triggered changes in the stability and secondary structure of a tetrameric dihydropyrimidinase: A biophysical characterization

Authors
Journal
Biophysical Chemistry
0301-4622
Publisher
Elsevier
Publication Date
Volume
139
Issue
1
Identifiers
DOI: 10.1016/j.bpc.2008.10.003
Keywords
  • Metalloenzyme
  • Protein Structure And Stability
  • Fluorescence
  • Circular Dichroism
  • Zinc
  • Molten Globule
Disciplines
  • Biology
  • Chemistry

Abstract

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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