Serine-rich repeat glycoproteins (SRRPs) are a growing family of bacterial adhesins found in many streptococci and staphylococci; they play important roles in bacterial biofilm formation and pathogenesis. Glycosylation of this family of adhesins is essential for their biogenesis. A glucosyltransferase (Gtf3) catalyzes the second step of glycosylation of a SRRP (Fap1) from an oral streptococcus, Streptococcus parasanguinis. Although Gtf3 homologs are highly conserved in SRRP-containing streptococci, they share minimal homology with functionally known glycosyltransferases. We report here the 2.3 Å crystal structure of Gtf3. The structural analysis indicates that Gtf3 forms a tetramer and shares significant structural homology with glycosyltransferases from GT4, GT5, and GT20 subfamilies. Combining crystal structural analysis with site-directed mutagenesis and in vitro glycosyltransferase assays, we identified residues that are required for UDP-or UDP-glucose binding and for oligomerization of Gtf3 and determined their contribution to the enzymatic activity of Gtf3. Further in vivo studies revealed that the critical amino acid residues identified by the structural analysis are crucial for Fap1 glycosylation in S. parasanguinis in vivo. Moreover, Gtf3 homologs from other streptococci were able to rescue the gtf3 knock-out mutant of S. parasanguinis in vivo and catalyze the sugar transfer to the modified SRRP substrate in vitro, demonstrating the importance and conservation of the Gtf3 homologs in glycosylation of SRRPs. As the Gtf3 homologs only exist in SRRP-containing streptococci, we conclude that the Gtf3 homologs represent a unique subfamily of glycosyltransferases.Serine-rich repeat glycoproteins (SRRPs) 2 are a growing family of bacterial adhesins found in many streptococci, staphylococci, and other Gram-positive bacteria (1). They have been shown to be important for bacterial biofilm formation and pathogenesis. A number of SRRPs including GspB (2) and Hsa (3) of Streptococcus gordonii and SraP of Staphylococcus aureus (4) have been implicated in pathogenesis of infective endocarditis. This family of streptococcal adhesins also mediates other infectious processes. PsrP is associated with pathogenesis of invasive pneumococcal disease by Streptococcus pneumoniae (5). Srr-1 is involved in crossing the blood-brain barrier by Streptococcus agalactiae to induce meningitis (6); Srr-2 is required for neonatal infection by S. agalactiae (7). Furthermore, vaccination against Srr-2 prevents maternal disease transfer caused by S. agalactiae COH1 in a neonatal animal model (8); immunization of experimental animals by PsrP prevents invasive pneumonia induced by S. pneumoniae in a mouse model (5).Fimbriae-associated protein 1 (Fap1) from an oral streptococcus, Streptococcus parasanguinis, was the first SRRP identified (9, 10). As its biological function and biosynthetic pathways have been studied extensively (1), Fap1 has emerged as a model to investigate the SRRP family of bacterial adhesins (11). Fap1 is required for fimb...