The family 52 beta-xylosidase from Geobacillus stearothermophilus is a dimer: structural and biophysical characterization of a glycoside hydrolase.
Xylans are the most abundant polysaccharides forming the plant cell wall hemicelluloses, and they are degraded, among other proteins, by beta-xylosidase enzymes. In this work, the structural and biophysical properties of the family 52 beta-xylosidase from Geobacillus stearothermophilus, XynB2, are described. Size exclusion chromatography, analytical centrifugation, ITC, CD, fluorescence (steady state and ANS-binding) and FTIR were used to obtain the structure, the oligomerization state and the conformational changes of XynB2, as pH, chemical denaturants or temperature were modified. This report describes the first extensive conformational characterization of a family 52 beta-xylosidase. The active protein was a highly hydrated dimer, whose active site was formed by the two protomers, and it probably involved aromatic residues. At low pH, the protein was not active and it populated a monomeric molten-globule-like species, which had a conformational transition with a pK(a) of approximately 4.0. Thermal and chemical-denaturations of the native protein showed hysteresis behaviour. The protein at physiological pH was formed by alpha-helix (30%) and beta-sheet (30%), as shown by CD and FTIR. Comparison with other xylosidases of the same family indicates that the percentages of secondary structure seem to be conserved among the members of the family.
Contreras
Lellys M
LM
Departamento de Biología, Facultad Experimental de Ciencias y Tecnología, Universidad de Carabobo, 2001 Valencia, Venezuela. contrera@uc.edu.ve
Gómez
Javier
J
Prieto
Jesús
J
Clemente-Jiménez
Josefa M
JM
Las Heras-Vázquez
Francisco J
FJ
Rodríguez-Vico
Felipe
F
Blanco
Francisco J
FJ
Neira
José L
JL
D001406:Bacillaceae / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry*; D002942:Circular Dichroism / Q000379:methods; D019281:Dimerization; D043364:Endo-1,4-beta Xylanases / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D020836:Protein Structure, Quaternary / Q000502:physiology; D017433:Protein Structure, Secondary / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods
D001426:Bacterial Proteins; D043364:Endo-1,4-beta Xylanases