Proteome- wide bioinformatic annotation and functional validation of the monotopic phosphoglycosyl transferase superfamily

• 成果类型: Article
• 署名作者: Durand, Theo; Dodge, Greg J.; Siuda, Roxanne P.; Higinbotham, Hugh R.; Arbour, Christine A.; Ghosh, Soumi; Allen, Karen N.; Imperiali, Barbara
• 署名单位: Massachusetts Institute of Technology (MIT); Massachusetts Institute of Technology (MIT); Imperial College London; Biogen; Boston University; Boston University
• 刊物名称: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• ISSN/ISSBN: 0027-10406
• DOI: 10.1073/pnas.2417572121
• 发表日期: 2024-12-03
• 关键词: acetyl-d-quinovosamine; in-vitro biosynthesis; capsular polysaccharide; catalytic mechanism; o-antigen; lipopolysaccharide; carbohydrate; CONSERVATION; 1-phosphate; fucosamine

摘要

Phosphoglycosyl transferases (PGTs) are membrane proteins that initiate glycoconjugate biosynthesis by transferring a phospho-sugar moiety from a soluble nucleoside diphosphate sugar to a membrane- embedded polyprenol phosphate acceptor. The centrality of PGTs in complex glycan assembly and the current lack of functional information make these enzymes high- value targets for biochemical investigation. In particular, the small monotopic PGT family is exclusively bacterial and represents the minimal functional unit of the monotopic PGT superfamily. Here, we combine a sequence similarity network analysis with a generalizable, luminescence- based activity assay to probe the substrate specificity of this family of monoPGTs in the bacterial cell- membrane fraction. This strategy allows us to identify specificity on a far more significant scale than previously achievable and correlate preferred substrate specificities with predicted structural differences within the conserved monoPGT fold. Finally, we present the proof- of- concept for a small- scale inhibitor screen (eight nucleoside analogs) with four monoPGTs of diverse substrate specificity, thus building a foundation for future inhibitor discovery initiatives.