Department of Biology and Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139.
Imperial College London, South Kensington, London SW7 2AZ, United Kingdom.
Proc Natl Acad Sci U S A. 2024 Dec 3;121(49):e2417572121. doi: 10.1073/pnas.2417572121. Epub 2024 Nov 27.
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.
磷酸糖基转移酶（PGTs）是一种膜蛋白，通过将磷酸糖部分从可溶性核苷二磷酸糖转移到膜嵌入的多萜醇磷酸受体上，启动糖缀合物的生物合成。PGTs 在复杂聚糖组装中的核心地位以及当前缺乏功能信息，使这些酶成为生化研究的高价值目标。特别是，小的单拓扑 PGT 家族是细菌特有的，代表了单拓扑 PGT 超家族的最小功能单元。在这里，我们结合序列相似性网络分析和可推广的基于发光的活性测定，在细菌细胞膜部分中探测这个单 PGTS 家族的底物特异性。这种策略使我们能够在比以前更显著的规模上识别特异性，并将首选的底物特异性与保守的单 PGTS 折叠内预测的结构差异相关联。最后，我们提出了一个小规模抑制剂筛选（8 种核苷类似物）的概念验证，针对具有不同底物特异性的四个单 PGTS，从而为未来的抑制剂发现计划奠定了基础。
