IDENTIFICATION OF ACTIVE SITE RESIDUES OF THE INVERTING GLYCOSYLTRANSFERASE CGS REQUIRED FOR THE SYNTHESIS OF CYCLIC b-1,2-GLUCAN, A Brucella abortus VIRULENCE FACTOR

ANDRÉS E. CIOCCHINI, MARA S. ROSET, GABRIEL BRIONES, NORA IÑÓN DE IANNINO AND RODOLFO A. UGALDE

GLYCOBIOLOGY

Oxford University Press

Lugar: Oxford, Inglaterra; Año: 2006 vol. 16 p. 679 - 691

0959-6658

B. abortus cyclic glucan synthase (Cgs) is a 320-kDa (2,868-amino-acid) polytopic integral inner membrane protein responsible for the synthesis of the virulence factor cyclic b-1,2-glucan by a novel mechanism in which the enzyme itself acts as a protein intermediate. Cgs functions as an inverting processive b-1,2-autoglucosyltransferase, and has the three-enzymatic activities required for synthesis of the cyclic glucan: initiation, elongation, and cyclization. To gain further insight into the protein domains that are essential for the enzymatic activity, we have compared the Cgs sequence to other glycosyltransferases. This procedure allowed us to identify in the Cgs-region (475-818) the widely spaced D, DxD, E/D, (Q/R)xxRW motif that is highly conserved in the active site of numerous glycosyltransferases. By site-directed mutagenesis and in vitro and in vivo activity assays, we have demonstrated that most of the amino acid residues of this motif are essential for Cgs activity. These sequence and site-directed mutagenesis analysis also indicate that Cgs should be considered a bi-functional modular glycosyltransferase with an N-terminal glycosyltransferase domain belonging to a new GT family related to GT-2 (GT-84) followed by a GH-94 glycoside hydrolase C-terminal domain. Furthermore, over-expression of inactive mutants results in wild type production of cyclic glucan when bacteria co-express the mutant and the wild type form, indicating that Cgs may function in the membrane as a monomeric enzyme. Together, these results are compatible with a single addition model by which Cgs acts in the membrane as a monomer, and uses the identified motif to form a single center for substrate binding and glycosyl-transfer reaction.