Structure and conformational stability of a tetrameric thermostable N‐succinylamino acid racemase |
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Authors: | Joaquín Pozo‐Dengra Sergio Martínez‐Rodríguez Lellys M. Contreras Jesús Prieto Montserrat Andújar‐Sánchez Josefa M. Clemente‐Jiménez Francisco J. Las Heras‐Vázquez Felipe Rodríguez‐Vico José L. Neira |
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Affiliation: | 1. Departamento de Química Física, Bioquímica y Química Inorgánica, Universidad de Almería, 04120 Almería, Spain;2. Departamento de Biología, Facultad Experimental de Ciencias y Tecnología, Universidad de Carabobo, 2001 Valencia, Venezuela;3. Structural Biology and Biocomputing Programme, Centro Nacional de Investigaciones Oncológicas (CNIO), 28007 Madrid, Spain;4. Instituto de Biología Molecular y Celular, Universidad Miguel Hernández, 03202 Elche (Alicante), Spain;5. Biocomputation and Complex Systems Physics Institute, 50009 Zaragoza, Spain |
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Abstract: | The N‐succinylamino acid racemases (NSAAR) belong to the enolase superfamily and they are large homooctameric/hexameric species that require a divalent metal ion for activity. We describe the structure and stability of NSAAR from Geobacillus kaustophilus (GkNSAAR) in the absence and in the presence of Co2+ by using hydrodynamic and spectroscopic techniques. The Co2+, among other assayed divalent ions, provides the maximal enzymatic activity at physiological pH. The protein seems to be a tetramer with a rather elongated shape, as shown by AU experiments; this is further supported by the modeled structure, which keeps intact the largest tetrameric oligomerization interfaces observed in other homooctameric members of the family, but it does not maintain the octameric oligomerization interfaces. The native functional structure is mainly formed by α‐helix, as suggested by FTIR and CD deconvoluted spectra, with similar percentages of structure to those observed in other protomers of the enolase superfamily. At low pH, the protein populates a molten‐globule‐like conformation. The GdmCl denaturation occurs through a monomeric intermediate, and thermal denaturation experiments indicate a high thermostability. The presence of the cofactor Co2+ did alter slightly the secondary structure, but it did not modify substantially the stability of the protein. Thus, GkNSAAR is one of the few members of the enolase family whose conformational propensities and stability have been extensively characterized. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 757–772, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com |
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Keywords: | N‐succinylamino acid racemase protein stability protein structure tetramer fluorescence enolase superfamily |
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