Structure and conformational stability of a tetrameric thermostable N-succinylamino acid racemase

• Published in: Biopolymers Vol. 91; no. 9; pp. 757 - 772
• Main Authors: Pozo-Dengra, Joaquín, Martínez-Rodríguez, Sergio, Contreras, Lellys M., Prieto, Jesús, Andújar-Sánchez, Montserrat, Clemente-Jiménez, Josefa M., Las Heras-Vázquez, Francisco J., Rodríguez-Vico, Felipe, Neira, José L.
• Format: Journal Article
• Language: English
• Published: Hoboken Wiley Subscription Services, Inc., A Wiley Company 01.09.2009
• Subjects: Amino Acid Sequence; Amino Acids - chemistry; Amino Acids - metabolism; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Cobalt - chemistry; enolase superfamily; Enzyme Stability; fluorescence; Geobacillus; Hot Temperature; Hydrogen-Ion Concentration; Models, Molecular; Molecular Sequence Data; N-succinylamino acid racemase; Protein Conformation; Protein Denaturation; protein stability; protein structure; Racemases and Epimerases - chemistry; Racemases and Epimerases - genetics; Racemases and Epimerases - metabolism; Sequence Alignment; Spectroscopy, Fourier Transform Infrared; tetramer; Thermodynamics
• ISSN: 0006-3525; 1097-0282
• DOI: 10.1002/bip.21226

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