IDENTIFICATION OF ACTIVE SITE RESIDUES OF THE INVERTING GLYCOSYLTRANSFERASE Cgs FROM Brucella abortus

CIOCCHINI AE, ROSET MS, BRIONES G, IÑON DE IANNINO N, UGALDERA

Pinamar, Buenos Aires, Argentina

Congreso; XLI Reunion Anual de la Sociedad Argentina de Investigaciones en Bioquimica y Biologia Molecular (SAIB 2005).; 2005

Sociedad Argentina de Investigaciones en Bioquimica y Biologia Molecular (SAIB)

Brucella abortus cyclic glucan synthase (Cgs) is a 320-kD polytopic integral inner membrane protein responsible for the synthesis of cyclic b-1,2-glucan. To gain further insight into the protein domains essential for enzyme activity such as active site/s, we have compared the Cgs sequence to other glycosyltransferases, and we have identified the widely spaced D, DXD, E/D, (Q/R)XXRW motif that is highly conserved in all Cgs and 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 these residues are involved in the activity of Cgs. Cgs-(475-818) domain, where the D, DXD, D/E, (Q/R)XXRW motif was identified, may be implicated in [UDP-Glc:b-(1,2)oligosaccharide glucosyltransferase] activity being responsible for chain elongation during cyclic glucan synthesis. Furthermore, over-expression of inactive mutants results in wild type production of cyclic glucan when cells co-express the mutant form and the wild type form. Together, these results are compatible with a single addition model by which Cgs acts in the membrane as a monomeric enzyme, and uses the D, DXD, D/E, (Q/R)XXRW motif to form a single center for substrate binding and glycosyl transfer reaction.