PDF(Pubmed)
Abstract：
Bacteroidales (syn. Bacteroidetes) are prominent members of the human gastrointestinal ecosystem mainly due to their efficient glycan-degrading machinery, organized into gene clusters known as polysaccharide utilization loci (PULs). A single PUL was reported for catabolism of high-mannose (HM) N-glycan glyco-polypeptides in the gut symbiont Bacteroides thetaiotaomicron, encoding a surface endo-β-N-acetylglucosaminidase (ENGase), BT3987. Here, we discover an ENGase from the GH18 family in B. thetaiotaomicron, BT1285, encoded in a distinct PUL with its own repertoire of proteins for catabolism of the same HM N-glycan substrate as that of BT3987. We employ X-ray crystallography, electron microscopy, mass spectrometry-based activity measurements, alanine scanning mutagenesis and a broad range of biophysical methods to comprehensively define the molecular mechanism by which BT1285 recognizes and hydrolyzes HM N-glycans, revealing that the stabilities and activities of BT1285 and BT3987 were optimal in markedly different conditions. BT1285 exhibits significantly higher affinity and faster hydrolysis of poorly accessible HM N-glycans than does BT3987. We also find that two HM-processing endoglycosidases from the human gut-resident Alistipes finegoldii display condition-specific functional properties. Altogether, our data suggest that human gut microbes employ evolutionary strategies to express distinct ENGases in order to optimally metabolize the same N-glycan substrate in the gastroinstestinal tract.
摘要：
拟杆菌（同系物。拟杆菌）是人类胃肠道生态系统的重要成员，主要是由于其有效的聚糖降解机制，组织成称为多糖利用基因座(PULs)的基因簇。据报道，肠道共生体拟杆菌中高甘露糖（HM）N-聚糖糖多肽的分解代谢存在单个PUL，编码表面内切-β-N-乙酰氨基葡萄糖苷酶（ENGase），BT3987.这里,我们在B.thetaiotaomicron中发现了一个GH18家族的ENGase，BT1285，以独特的PUL编码，其自身的蛋白质库用于与BT3987相同的HMN-聚糖底物的分解代谢。我们使用X射线晶体学，电子显微镜，基于质谱的活性测量，丙氨酸扫描诱变和广泛的生物物理方法，以全面定义BT1285识别和水解HMN-聚糖的分子机制，表明BT1285和BT3987的稳定性和活性在明显不同的条件下是最佳的。BT1285表现出比BT3987显著更高的亲和力和不易接近的HMN-聚糖的更快水解。我们还发现，来自人类肠道定居的Alistipesfinegoldii的两种HM加工内切糖苷酶显示出特定条件的功能特性。总之,我们的数据表明,人类肠道微生物采用进化策略来表达不同的ENGase,以便在胃肠道中最佳地代谢相同的N-聚糖底物.
