Abstract
The accessory Sec system is a specialized transport system that exports serine-rich repeat (SRR) glycoproteins of Gram-positive bacteria. This system contains two homologues of the general secretory (Sec) pathway (SecA2 and SecY2) and several other essential proteins (Asp1 to Asp5) that share no homology to proteins of known function. In Streptococcus gordonii, Asp2 is required for the transport of the SRR adhesin GspB, but its role in export is unknown. Tertiary structure predictions suggest that the carboxyl terminus of Asp2 resembles the catalytic region of numerous enzymes that function through a Ser-Asp-His catalytic triad. Sequence alignment of all Asp2 homologues identified a highly conserved pentapeptide motif (Gly-X-Ser(362)-X-Gly) typical of most Ser-Asp-His catalytic triads, where Ser forms the reactive residue. Site-directed mutagenesis of residues comprising the predicted catalytic triad of Asp2 of S. gordonii had no effect upon GspB transport but did result in a marked change in the electrophoretic mobility of the protein. Lectin-binding studies and monosaccharide content analysis of this altered glycoform revealed an increase in glucosamine deposition. Random mutagenesis of the Asp2 region containing this catalytic domain also disrupted GspB transport. Collectively, our findings suggest that Asp2 is a bifunctional protein that is essential for both GspB transport and correct glycosylation. The catalytic domain may be responsible for controlling the glycosylation of GspB, while other surrounding regions are functionally required for glycoprotein transport.