环状β-1,2-葡聚糖合酶(CGS)是产生环状β-1,2-葡聚糖(CβG)的关键酶,该酶参与细菌感染或与宿主生物的共生。然而,环化机制,CGS反应的最后一步,还没有被完全理解。在这里,我们对意大利热厌氧杆菌(TiCGSCy)的CGS环化域进行了功能和结构分析。我们首先发现,TiCGSCy以线性β-1,2-葡聚糖为底物生产β-葡萄糖苷酶抗性化合物。1H-NMR分析显示这些产物是CβG。接下来,使用β-1,2-葡萄糖寡糖的作用模式分析揭示了独特的反应模式:不水解的专有转糖基化和六糖是底物的最小长度。这些分析还表明,较长的底物β-1,2-葡萄糖寡糖是优选的,与CGS通常产生聚合度约为20的CβG的事实一致。最后,发现TiCGSCy的环化结构域的整体结构与系统发育不同组中的β-1,2-葡聚糖酶的结构相似。同时,鉴定的催化残基表明这些酶之间的反应途径存在明显差异。总的来说,我们提出了一种新的TiCGSCy反应机理。因此,该组CGS定义了一个新的糖苷水解酶家族,GH189.关键词:•清楚地证明环化结构域单独产生环状β-1,2-葡聚糖。•结构域专门催化转糖基化而不水解。•本发明的催化结构域定义为新的糖苷水解酶家族189。
Cyclic β-1,2-glucan synthase (CGS) is a key enzyme in production of cyclic β-1,2-glucans (CβGs) which are involved in bacterial infection or symbiosis to host organisms. Nevertheless, a mechanism of cyclization, the final step in the CGS reaction, has not been fully understood. Here we performed functional and structural analyses of the cyclization domain of CGS alone from Thermoanaerobacter italicus (TiCGSCy). We first found that β-glucosidase-resistant compounds are produced by TiCGSCy with linear β-1,2-glucans as substrates. The 1H-NMR analysis revealed that these products are CβGs. Next, action pattern analyses using β-1,2-glucooligosaccharides revealed a unique reaction pattern: exclusive
transglycosylation without hydrolysis and a hexasaccharide being the minimum length of the substrate. These analyses also showed that longer substrate β-1,2-glucooligosaccharides are preferred, being consistent with the fact that CGSs generally produce CβGs with degrees of polymerization of around 20. Finally, the overall structure of the cyclization domain of TiCGSCy was found to be similar to those of β-1,2-glucanases in phylogenetically different groups. Meanwhile, the identified catalytic residues indicated clear differences in the reaction pathways between these enzymes. Overall, we propose a novel reaction mechanism of TiCGSCy. Thus, the present group of CGSs defines a new glycoside hydrolase family, GH189. KEY POINTS: • It was clearly evidenced that cyclization domain alone produces cyclic β-1,2-glucans. • The domain exclusively catalyzes
transglycosylation without hydrolysis. • The present catalytic domain defines as a new glycoside hydrolase family 189.