Identification of active site residues of the inverting glycosyltransferase Cgs required for the synthesis of cyclic beta-1,2-glucan, a Brucella abortus virulence factor.
Brucella abortus cyclic glucan synthase (Cgs) is a 320-kDa (2868-amino acid) polytopic integral inner membrane protein responsible for the synthesis of the virulence factor cyclic beta-1,2-glucan by a novel mechanism in which the enzyme itself acts as a protein intermediate. Cgs functions as an inverting processive beta-1,2-autoglucosyltransferase and has the three enzymatic activities required for the 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 with other glycosyltransferases (GTs). 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 GTs. 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 analyses also indicate that Cgs should be considered a bi-functional modular GT, with an N-terminal GT 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 (WT) production of cyclic glucan when bacteria co-express the mutant and the WT 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.
Ciocchini
Andrés E
AE
Instituto de Investigaciones Biotecnológicas-Instituto Tecnológico de Chascomús, CONICET-Universidad Nacional de General San Martín, Buenos Aires, Argentina.
Roset
Mara S
MS
Briones
Gabriel
G
Iñón de Iannino
Nora
N
Ugalde
Rodolfo A
RA
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D002003:Brucella abortus / Q000201:enzymology* / Q000472:pathogenicity; D017124:Conserved Sequence; D016695:Glycosyltransferases / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein; D037521:Virulence Factors / Q000737:chemistry* / Q000235:genetics; D047071:beta-Glucans / Q000378:metabolism*
D001426:Bacterial Proteins; D037521:Virulence Factors; D047071:beta-Glucans; C080135:cyclic beta(1-2)glucan; D016695:Glycosyltransferases