Functional mapping of Brucella abortus cyclic beta-1,2-glucan synthase: identification of the protein domain required for cyclization.

Cyclic beta-1,2-glucans (CbetaG) are periplasmic homopolysaccharides that have been shown to play an important role in several symbiotic and pathogenic relationships. Cyclic beta-1,2-glucan synthase (Cgs), the enzyme responsible for the synthesis of CbetaG, is an integral membrane polyfunctional protein that catalyzes the four enzymatic activities (initiation, elongation, phosphorolysis, and cyclization) required for the synthesis of CbetaG. Recently, we have identified the glycosyltransferase and the beta-1,2-glucooligosaccharide phosphorylase domains of Brucella abortus Cgs. In this study, we performed large-scale linker-scanning mutagenesis to gain further insight into the functional domains of Cgs. This analysis allowed us to construct a functional map of the enzyme and led to the identification of the minimal region required for the catalysis of initiation and elongation reactions. In addition, we identified the Cgs region (residues 991 to 1544) as being the protein domain required for cyclization and demonstrated that upon cyclization and releasing of the CbetaG, one or more glucose residues remain attached to the protein intermediate that serves as a primer for the next round of CbetaG synthesis. Finally, our results indicate that the overall control of the degree of polymerization of CbetaG is the result of a balance between elongation, phosphorolysis, and cyclization reactions.