Thermostable xylanase from Thermoascus aurantiacus at ultrahigh resolution (0.89 Å) at 100 K and atomic resolution (1.11 Å) at 293 K refined anisotropically to small-molecule accuracy

• Published in: Acta crystallographica. Section D, Biological crystallography. Vol. 59; no. 1; pp. 105 - 117
• Main Authors: Natesh, R., Manikandan, K., Bhanumoorthy, P., Viswamitra, M. A., Ramakumar, S.
• Format: Journal Article
• Language: English
• Published: 5 Abbey Square, Chester, Cheshire CH1 2HU, England Munksgaard International Publishers 01-01-2003
• Subjects: ACCURACY; Anisotropy; Arginine - chemistry; Ascomycota - enzymology; atomic resolution; BASIC BIOLOGICAL SCIENCES; Crystallography, X-Ray - methods; Enzyme Stability; Glutamic Acid - chemistry; Hydrogen Bonding; Models, Molecular; NATIONAL SYNCHROTRON LIGHT SOURCE; plasticity; Protein Folding; Protein Structure, Secondary; RESOLUTION; Temperature; Water - chemistry; Xylan Endo-1,3-beta-Xylosidase; XYLANASE; xylanases; Xylosidases - chemistry
• ISSN: 1399-0047; 0907-4449; 1399-0047
• DOI: 10.1107/S0907444902020164

Thermoascus aurantiacus xylanase is a thermostable enzyme which hydrolyses xylan, a major hemicellulose component of the biosphere. The crystal structure of this F/10 family xylanase, which has a triosephosphate isomerase (TIM) barrel (β/α)8 fold, has been solved to small‐molecule accuracy at atomic resolution (1.11 Å) at 293 K (RTUX) and at ultrahigh resolution (0.89 Å) at 100 K (CTUX) using X‐ray diffraction data sets collected on a synchrotron light source, resulting in R/Rfree values of 9.94/12.36 and 9.00/10.61% (for all data), respectively. Both structures were refined with anisotropic atomic displacement parameters. The 0.89 Å structure, with 177 476 observed unique reflections, was refined without any stereochemical restraints during the final stages. The salt bridge between Arg124 and Glu232, which is bidentate in RTUX, is water‐mediated in CTUX, suggesting the possibility of plasticity of ion pairs in proteins, with water molecules mediating some of the alternate arrangements. Two buried waters present inside the barrel form hydrogen‐bond interactions with residues in strands β2, β3, β4 and β7 and presumably contribute to structural stability. The availability of accurate structural information at two different temperatures enabled the study of the temperature‐dependent deformations of the TIM‐barrel fold of the xylanase. Analysis of the deviation of corresponding Cα atoms between RTUX and CTUX suggests that the interior β‐strands are less susceptible to changes as a function of temperature than are the α‐helices, which are on the outside of the barrel. βα‐loops, which are longer and contribute residues to the active‐site region, are more flexible than αβ‐loops. The 0.89 Å structure represents one of the highest resolution structures of a protein of such size with one monomer molecule in the asymmetric unit and also represents the highest resolution TIM‐barrel fold structure to date. It may provide a useful template for theoretical modelling studies of the structure and dynamics of the ubiquitous TIM‐barrel fold.