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

GUIDOLIN, L. SOLEDAD *; CIOCCHINI, ANDRÉS E. *; IÑÓN DE IANNINO, NORA; UGALDE, RODOLFO A.; * GUIDOLIN, L. SOLEDAD Y CIOCCHINI, ANDRES E. COLABORARON IGUALMENTE EN LA REALIZACION DE ESTE TRABAJO.

JOURNAL OF BACTERIOLOGY

American Society for Microbiology

Lugar: Washington, USA; Año: 2009 vol. 191 p. 1230 - 1238

0021-9193

Cyclic beta-1,2-glucans (CBG) 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 CBG, is an integral membrane polyfunctional protein that catalyzes the four enzymatic activities (initiation, elongation, phosphorolysis and cyclization) required for the synthesis of CBG. 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 catalysis of initiation and elongation reactions. In addition, we identified the Cgs-region (991-1544) as the protein domain required for cyclization and demonstrated that upon cyclization and releasing of the CBG one or more glucose residues remain attached to the protein intermediate that serves as a primer for the next round of CBG synthesis. Finally, our results indicate that the overall control of CBG degree of polymerization is the result of a balance between elongation, phosphorolysis and cyclization reactions.