Molecular and structural biostudies on streptococcalinyasion/infection mechanisms to host cells through degradation of extracellular matrices glyoceaminoglycans

一些病原微生物产生多糖降解酶(裂解酶和水解酶)来入侵哺乳动物和/或植物细胞。哺乳动物糖胺多聚糖构成细胞表面基质的一部分，是微生物酶的典型靶标。不饱和葡萄糖醛酸水解酶(UGL)，首次在芽孢杆菌中发现。菌株GL1催化通过降解基质的多糖裂解酶(如透明质酸和软骨素裂解酶)的反应从寡糖中水解性释放不饱和糖醛酸，这表明这些酶在微生物感染中起着毒力因子的作用。本研究分析了糖胺多糖降解酶裂解酶和水解酶的结构和功能关系，结合酶-底物复合体的X射线结晶学和定点诱变，阐明了透明质酸裂解酶同系物(黄原胶裂解酶)的催化反应和底物识别机理。单一酪氨酸…更多的残基提取+1位尿酸残基的C5-质子，并将质子贡献给糖苷键进行裂解。与一般糖苷水解酶具有异常构型的保留或反转催化机制不同，UGL独特地触发不饱和尿酸残基中的乙烯基醚基团的水解，而不是糖苷键的水解。最近的全基因组序列分析表明，从细菌到真菌(超过70种)的大量微生物的基因组中都有UGL同源基因。在碳水化合物活性酶(CAZy)数据库中，UGL及其同源物形成了一个新的家族，GH-88。UGL的微生物生产者包括梭状芽胞杆菌、链球菌和弧菌等病原菌。该酶的同源基因在链球菌、无乳链球菌、肺炎链球菌、化脓性链球菌和猪链球菌中被发现。与芽孢杆菌UGL不同，链球菌UGL作用于不饱和软骨素二糖，在GalNAc的C4处有一个硫酸盐基团。较少

Some pathogenic microorganisms produce polysaccharide-degrading enzymes (lyases and hydrolases) to invade mammalian and/or plant cells. Mammalian glycosaminoglycans that form part of cell surface matrix are typical targets for microbial enzymes. Unsaturated glucuronyl hydrolase (UGL), which was genetically first identified in Bacillus sp. strain GL1, catalyzes the hydrolytic release of an unsaturated uronic acid from oligosaccharides produced through the reaction of the matrix-degrading polysaccharide lyases (e.g., hyaluronate and chondroitin lyases), suggesting that these enzymes function as a virulent factor in microbial infection. In this study, structure and function relationship of glycosaminoglycan-degrading enzymes, lyase and hydrolase, was analyzed.Based on X-ray crystallography of enzyme-substrate complexes and site-directed mutagenesis, mechanisms for catalytic reaction and substrate recognition of hyaluronate lyase homologue (xanthan lyase) were clarified. A single tyrosine … More residue abstracts C5-proton of the uronate residue at subsite +1 and donates the proton to glycosidic bond to be cleaved.In contrast with general glycoside hydrolases with the retention or inversion catalytic mechanism of an anomeric configuration, UGL uniquely triggers hydrolysis of vinyl ether groups in the unsaturated uronate residue but not of the glycosidic bond.Recent complete genome sequence analyses indicate that a large number of microorganisms ranging from bacteria to fungi (over 70 species) have a UGL homologous gene in their genome. In the Carbohydrate-Active enZyme (CAZy) database, UGL and its homologues form a new family, GH-88. Microbial producers of UGL include pathogenic bacteria such as clostridia, streptococci, and vibrios. The enzyme homologous gene has been found especially in streptococci, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes, and Streptococcus suis. Unlike the bacillus UGL, streptococcal UGL acts on unsaturated chondroitin disaccharide with a sulfate group at C4 of GalNAc. Less