Natesh R, Manikandan K, Bhanumoorthy P, Viswamitra M A, Ramakumar S
Department of Physics, Indian Institute of Science, Bangalore 560 012, India.
Acta Crystallogr D Biol Crystallogr. 2003 Jan;59(Pt 1):105-17. doi: 10.1107/s0907444902020164. Epub 2002 Dec 19.
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 (beta/alpha)(8) fold, has been solved to small-molecule accuracy at atomic resolution (1.11 A) at 293 K (RTUX) and at ultrahigh resolution (0.89 A) at 100 K (CTUX) using X-ray diffraction data sets collected on a synchrotron light source, resulting in R/R(free) 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 A 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 beta2, beta3, beta4 and beta7 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 C(alpha) atoms between RTUX and CTUX suggests that the interior beta-strands are less susceptible to changes as a function of temperature than are the alpha-helices, which are on the outside of the barrel. betaalpha-loops, which are longer and contribute residues to the active-site region, are more flexible than alphabeta-loops. The 0.89 A 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.
嗜热栖热放线菌木聚糖酶是一种热稳定酶，可水解木聚糖，木聚糖是生物圈中主要的半纤维素成分。这种F/10家族木聚糖酶具有磷酸丙糖异构酶（TIM）桶状（β/α）8折叠结构，利用在同步辐射光源上收集的X射线衍射数据集，已分别在293 K（RTUX）下以原子分辨率（1.11 Å）和在100 K（CTUX）下以超高分辨率（0.89 Å）解析到小分子精度，R/R（自由）值分别为9.94/12.36和9.00/10.61%（针对所有数据）。两种结构均使用各向异性原子位移参数进行了精修。0.89 Å结构有177476个观测到的独特反射，在最后阶段精修时没有任何立体化学限制。在RTUX中为双齿的Arg124和Glu232之间的盐桥在CTUX中由水介导，这表明蛋白质中离子对具有可塑性的可能性，水分子介导了一些替代排列。桶状结构内部存在的两个埋藏水与β2、β3、β4和β7链中的残基形成氢键相互作用，大概有助于结构稳定性。在两个不同温度下获得的准确结构信息使得能够研究木聚糖酶TIM桶状折叠的温度依赖性变形。RTUX和CTUX之间相应Cα原子偏差的分析表明，内部β链比桶状结构外部的α螺旋对温度变化的敏感性更低。βα环更长且为活性位点区域贡献残基，比αβ环更灵活。0.89 Å结构代表了具有一个不对称单元中一个单体分子的这种大小蛋白质的最高分辨率结构之一，也是迄今为止最高分辨率的TIM桶状折叠结构。它可能为普遍存在的TIM桶状折叠的结构和动力学的理论建模研究提供有用的模板。
