Functional and structural analysis of a cyclization domain in a cyclic β-1,2-glucan synthase

Abstract

AbstractCyclic 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 fromThermoanaerobacter italicus(TiCGSCy). We first found that β-glucosidase-resistant compounds are produced by TiCGSCywith linear β-1,2-glucans as substrates. The1H-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 as the minimum length of the substrate. Nevertheless, 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 TiCGSCywas found to be similar to those of β-1,2-glucanases in phylogenetically different groups. Meanwhile, the identified catalytic residues indicated clear difference in the reaction pathways between these enzymes. Therefore, we propose a novel reaction mechanism of TiCGSCy. Thus, we propose that the present group of CGSs should be defined as a new glycoside hydrolase family, GHxxx.Statements and DeclarationsCompeting Interests: The authors declare no competing interests.Key PointsIt was clearly evidenced that cyclization domain alone produces cyclic β-1,2-glucans.The domain exclusively catalyzes transglycosylation without hydrolysis.The present catalytic domain should be defined as a new glycoside hydrolase family.