title: Structure and conformational stability of a tetrameric thermostable N-succinylamino acid racemase.
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 Co(2+) by using hydrodynamic and spectroscopic techniques. The Co(2+), 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 alpha-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 Co(2+) 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.
authors.affiliation: Departamento de Química Física, Bioquímica y Química Inorgánica, Universidad de Almería, 04120 Almería, Spain.
authors.forename: Joaquín
authors.initials: J
authors.lastname: Pozo-Dengra
authors.forename: Sergio
authors.initials: S
authors.lastname: Martínez-Rodríguez
authors.forename: Lellys M
authors.initials: LM
authors.lastname: Contreras
authors.forename: Jesús
authors.initials: J
authors.lastname: Prieto
authors.forename: Montserrat
authors.initials: M
authors.lastname: Andújar-Sánchez
authors.forename: Josefa M
authors.initials: JM
authors.lastname: Clemente-Jiménez
authors.forename: Francisco J
authors.initials: FJ
authors.lastname: Las Heras-Vázquez
authors.forename: Felipe
authors.initials: F
authors.lastname: Rodríguez-Vico
authors.forename: José L
authors.initials: JL
authors.lastname: Neira
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003035:Cobalt / Q000737:chemistry; D004795:Enzyme Stability; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011489:Protein Denaturation; D019998:Racemases and Epimerases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017550:Spectroscopy, Fourier Transform Infrared; D013816:Thermodynamics
chemical_list: D000596:Amino Acids; D001426:Bacterial Proteins; D003035:Cobalt; D019998:Racemases and Epimerases