Structural and Functional Characterization of Phosphoglycosyl Transferases from Human Pathogens

Bacterial virulence and viability are strongly dependent on extracellular glycoconjugates including lipopolysaccharide (LPS), capsular polysaccharide (CPS), wall teichoic acid (WTA) and glycoproteins. While these glycoconjugates are diverse in structure and function, they share a common biosynthetic logic. For example, in Gram‐negative bacteria the biosynthesis of a variety of glycoconjugates are initiated on the cytoplasmic face of the bacterial inner membrane, with a phosphoglycosyl transferase (PGT) catalyzing the first membrane‐committed step of the pathway. PGTs transfer a phosphosugar from a nucleotide‐activated donor substrate onto a polyprenol phosphate such as undecaprenol phosphate (UndP). PGTs comprise two distinct superfamilies – polytopic, with multiple membrane spanning helices, and the more recently described monotopic, which contain an unusual re‐entrant membrane intercalating helix. To date, monotopic PGTs have only been observed in bacteria, and remain underexplored antimicrobial targets. In an effort to understand NDP‐sugar specificity and inform inhibitor design, a library of monotopic PGTs from human pathogens was expressed, purified, and subjected to structural and functional characterization. Both detergent and styrene maleic acid (SMA) copolymers were assessed for solubilization towards the purification and stabilization of these challenging protein targets. These efforts have led to purified samples of diverse monotopic PGTs for activity analyses using the UMP‐Glo ™ assay and crystallization screens, which have afforded reproduceable crystals for further investigation. Support or Funding Information R01GM131627, F32GM134576