Department of Pediatric Dentistry and Microbiology, University of Alabama at Birmingham, Schools of Dentistry and Medicine, Birmingham, Alabama 35294, USA.
J Biol Chem. 2011 Jul 29;286(30):27048-57. doi: 10.1074/jbc.M110.208629. Epub 2011 Jun 7.
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.
富含丝氨酸重复糖蛋白（SRRPs）是一类在许多链球菌和葡萄球菌中发现的不断增长的细菌黏附素家族；它们在细菌生物膜形成和发病机制中发挥重要作用。该黏附素家族的糖基化对于它们的生物发生至关重要。葡糖基转移酶（Gtf3）催化口腔链球菌副血链球菌中 SRRP（Fap1）糖基化的第二步。尽管 Gtf3 同源物在含 SRRP 的链球菌中高度保守，但它们与功能已知的糖基转移酶仅有最小的同源性。我们在此报告 Gtf3 的 2.3Å 晶体结构。结构分析表明，Gtf3 形成四聚体，并与 GT4、GT5 和 GT20 亚家族的糖基转移酶具有显著的结构同源性。结合晶体结构分析、定点突变和体外糖基转移酶测定，我们鉴定出了 Gtf3 结合 UDP 或 UDP-葡萄糖以及寡聚化所必需的残基，并确定了它们对 Gtf3 酶活性的贡献。进一步的体内研究表明，结构分析确定的关键氨基酸残基对于副血链球菌中 Fap1 的体内糖基化至关重要。此外，其他链球菌中的 Gtf3 同源物能够在体内拯救副血链球菌的 gtf3 敲除突变体，并在体外催化糖基转移到修饰的 SRRP 底物上，这证明了 Gtf3 同源物在 SRRPs 糖基化中的重要性和保守性。由于 Gtf3 同源物仅存在于含 SRRP 的链球菌中，我们得出结论，Gtf3 同源物代表了糖基转移酶的一个独特亚家族。
