IDENTIFICATION OF ESSENTIAL AMINO ACID RESIDUES IN THE BRUCELLA ABORTUS CYCLIC GLUCAN SYNTHASE CGS

CIOCCHINI, ANDRES E.; BRIONES, GABRIEL; IÑON DE IANNINO, NORA;UGALDE, RODOLFO A.

San Carlos de Bariloche, Rio Negro, Argentina

Congreso; XXXIX Reunion Anual de la Sociedad Argentina de Investigaciones en Bioquimica y Biologia Molecular (SAIB 2003); 2003

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

Cgs is an integral inner membrane protein of 316 kDa involved in the synthesis of cyclic b-(1,2)-glucan by a novel mechanism in which the enzyme acts itself as protein intermediate. Cgs uses UDP-glucose as donor-sugar and has the three-enzymatic activities required for the synthesis of the polysaccharide: initiation, elongation and cyclization. The first one may catalyze the transfer of the first glucose from UDP-Glc to an unknown amino acid of the protein intermediate. The second enzymatic activity [UDP-Glc:b-(1,2) oligosaccharide glucosyltransferase] may be responsible for chain elongation. Finally, the third activity may catalyze glucan cyclization and release from the protein. By comparing the sequence of Cgs to those of the members of glycosyltransferase family 2 (GTF2), we identified theD1, D2, D3, (Q/R)XXRW motif that is highly conserved in Cgs and in the putative active site of numerous processive b-glycosyltransferases. By site-directed mutagenesis we found that replacement of Asp-636 (D2) by asparagine abolished the enzyme activity in vitro and in vivo. Single substitutions of each of the amino acids of the RXXRW motif at positions 782, 785 and 786 with alanine resulted in the loss of enzyme activity in vitro. In vivo production of cyclic glucan was affected in the R785A and W786A mutants but not in the R782A mutant. These results indicate that Asp-636, Arg-785 and Trp-786 are essential for Cgs activity and may be implicated in its elongation activity.