Crystal structure at 1.8 Å resolution and proposed amino acid sequence of a thermostable xylanase from Thermoascus aurantiacus
Crystal structure at 1.8 Å resolution and proposed amino acid sequence of a thermostable xylanase from Thermoascus aurantiacus
复制标题
DOI：
10.1006/jmbi.1999.2727
复制
发表时间：
1999-05-21
影响因子：
5.6
通讯作者：
Viswamitra, MA
中科院分区：
文献类型：
--
作者：
Natesh, R;Bhanumoorthy, P;Viswamitra, MA
橙色嗜热菌木聚糖酶是一种耐热酶，它能水解生物圈中主要的半纤维素成分木聚糖。生长了属于P2（1）空间群的晶体，其中a = 41.7埃，B = 68.1埃，c = 51.4埃，β = 113.6度，Z = 2，其可以显示优于1.8埃的分辨率。采用变铅青链霉菌木聚糖酶模型，用分子置换法解析其结构。氨基酸序列由相关木聚糖酶序列的多重比对辅助的电子密度图确定。由此获得的序列提供了对先前基于生物化学方法报道的序列的校正。在1.8埃分辨率下具有301个氨基酸残基和266个水分子的最终精制蛋白质模型具有16.0%的R因子和21.1%的游离R，具有良好的立体化学。单链多肽呈现（α/β）（8）TIM-桶折叠，属于糖苷水解酶的F/10家族。活性位点由位于β-桶的C末端的两个谷氨酸残基组成，符合酶作用的双置换机制。一个二硫键和十多个盐桥已被确定。特别地，几乎被掩埋的盐桥Arg 124-Glu 232桥接催化谷氨酸残基所在的β链β 4和β 7，并且它可能在高温下的稳定性和活性中起关键作用。据我们所知，在F/10家族木聚糖酶中，我们第一次观察到α-螺旋α 6中间的脯氨酸残基，这可能有助于更好的包装。早期的研究表明，这种酶即使在70摄氏度下也能保持活性。改进的蛋白质模型允许与F/10酶家族中的其他已知结构进行详细比较。讨论了影响热稳定性的可能因素。(C)北京：科学出版社.
Thermoascus aurantiacus xylanase is a thermostable enzyme which hydrolyses xylan, a major hemicellulose component in the biosphere. Crystals belonging to P2(1) space group with a = 41.7 Angstrom, b = 68.1 Angstrom c = 51.4 Angstrom and beta = 113.6 degrees, Z = 2 were grown that could diffract to better than 1.8 Angstrom resolution. The structure was solved by molecular replacement method using the Streptomyces lividans xylanase model. The amino acid sequence was determined from the electron density map aided by multiple alignment of related xylanase sequences. The sequence thus obtained provides a correction to the sequence reported earlier based on biochemical methods. The final refined protein model at 1.8 Angstrom resolution with 301 amino acid residues and 266 water molecules has an R-factor of 16.0 % and free R of 21.1% with good stereochemistry. The single polypeptide chain assumes (alpha/beta)(8) TIM-barrel fold and belongs to F/10 family of glycoside hydrolases. The active site consists of two glutamate residues located at the C terminus end of the beta-barrel, conforming to the double displacement mechanism for the enzyme action. A disulphide bond and more than ten salt bridges have been identified. In particular, the salt bridge Arg124-Glu232 which is almost buried, bridges the beta-strands beta 4 and beta 7 where the catalytic glutamate residues reside, and it may play a key role in the stability and activity at elevated temperature. To our knowledge, for the first time in the F/10 family xylanases, we observe a proline residue in the middle of the alpha-helix alpha 6 which may be contributing to better packing. Earlier studies show that the enzyme retains its activity even at 70 degrees C. The refined protein model has allowed a detailed comparison with the other known structures in the F/10 family of enzymes. The possible causative factors for thermostability are discussed. (C) 1999 Academic Press.