MODELING XYLANASE SUBSTRATE SPECIFICITY

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 鉴于半纤维素是植物细胞壁中含量第二丰富的多糖，因此将半纤维素降解为组成糖并进而降解为乙醇是生物燃料生产中的关键步骤。木聚糖是木糖的1，4-或1，3-连接聚合物，可以用各种糖枝装饰。木聚糖酶(内切-1，4-木聚糖酶)通过双置换反应水解1，4-木聚糖的内部糖苷键，同时保留糖元糖的异常构型。然而，这些酶如何通过其V型底物结合裂隙与相对线性的结构结合仍有待完全了解。这个项目的目的可以概括为： 目的1：利用分子对接和分子动力学模拟，结合现有的结晶学数据，确定木聚糖酶底物结合裂解的末端区域如何有助于将聚合物底物呈递到这些酶的活性部位。 目的2：对底物-酶复合体进行计算能量分解，以确定底物结合裂隙的区域，这些区域可以被设计成增加对分支底物的识别，或者在木聚糖酶的糖锥区域引入新的亚位点来提高催化效率。 目的3：对目标1和目标2中预测的关键残基位置进行定点和高通量饱和突变，并评估后续克隆增强糖苷酶活性和底物特异性。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Degradation of hemicellulose to constituent sugars and consequently into ethanol is a key step in biofuel production, given that hemicellulose is the second most abundant polysaccharide in the plant cell wall. Xylans are ¿1,4- or ¿1,3-linked polymers of xylopyranose that can be found decorated with various sugar branches. Xylanases (endo-¿1,4-xylanases) hydrolyze the internal glycosidic bonds of ¿1,4 xylans through a double displacement reaction with retention of the anomeric configuration of the glycone sugar. How these enzymes bind to the relatively linear structure with their V-shaped substrate binding clefts however remains to be understood completely. The aim of this project can be summarized as: Aim 1: Use molecular docking and molecular dynamics simulations, together with existing crystallographic data, to determine how the distal regions of the substrate binding cleft of Xylanase contributes to the presentation of polymeric substrates into the active site of these enzymes. Aim 2: Perform computational energy decomposition on substrate-enzyme complexes to identify regions of the substrate binding cleft that can be engineered to increase recognition of branched substrates, or to introduce novel subsites into the glycone region of Xylanases to increase catalytic efficiency. Aim 3: Perform site-directed and high-throughput saturation, mutagenesis of the key residue positions, predicted in Aims 1 and 2, and evaluate the subsequent clones for enhanced glycosidase activity and substrate specificity.