Probing Monotopic Phosphoglycosyl Transferases from Complex Cellular Milieu

Abstract

Monotopic phosphoglycosyl transferase enzymes (monoPGTs) initiate the assembly of prokaryotic glycoconjugates essential for bacterial survival and proliferation. MonoPGTs belong to an expansive superfamily with a diverse and richly annotated sequence space; however, the biochemical roles of most monoPGTs in glycoconjugate biosynthesis pathways remain elusive. To better understand these critical enzymes, we have implemented activity-based protein profiling (ABPP) probes as protein-centric, membrane protein compatible tools that lay the groundwork for understanding the activity and regulation of the monoPGT superfamily from a cellular proteome. With straightforward gel-based readouts, we demonstrate robust, covalent labeling at the active site of various representative monoPGTs from cell membrane fractions using 3-phenyl-2H-azirine probes.

Figure 1.

Phosphoglycosyl transferases (PGTs) catalyze the initial membrane-committed step in the en bloc mechanism of glycoconjugate biosynthesis. The PrenPP-sugar product of the PGT reaction is further elaborated via sequential modification by glycosyltransferases (GTs) to afford PrenPP-glycoconjugates, that are translocated to the periplasmic space by a flippase. These membrane-bound glycoconjugates then continue on support essential functions related to bacterial survival and proliferation.

Figure 2

A) Ribbon diagram of monoPGT PglC (PDB 5W7L), inset and red coloring shows close-up view of active site highlighting the PglC Asp-Glu catalytic dyad. Sequence logo showing conservation in the catalytic residues among PglC orthologs. Logo generated using https://weblogo.berkeley.edu/logo.cgi. B) Bi-Bi ping-pong mechanism between a UDP-N,N'-diacetylbacillosamine (UDP-diNAcBac) as the UDP-sugar donor, and undecaprenol phosphate (Und-P) as the Pren-P acceptor. Proposed roles for binding are shown and catalytic residues are highlighted in red (numbering from Campylobacter concisus PglC).

Figure 3.

A) Synthesis the sulfonamide linker, and synthesis of probes Az-1 and Az-2. B) Western blot analysis of CEF containing overexpressed PglC-His₆ (Cj) in BL21, compared to CEF without any overexpressed proteins. The left most Western blot shows the fluorescence read out from the streptavidin fluorophore conjugate, and the middle shows the same Western blot imaged with Anti-His₆ fluorophore conjugate. The right most is a gel with the total protein stained by Coomassie. C) Probe labeling of His₆-SUMO-PglC (Cc) active site variants with Az-1 (100 μM). D) Probe labeling with Az-1 in a concentration dependent manner, followed by a click reaction with monodispersed N₃-PEG₃₆, demonstrating a single covalent modification visualized with a single mass shift.

Figure 4

Western blot analysis of CEF containing overexpressed PglC-His₆ in BL21, compared to CEF without any overexpressed proteins. The left most Western blot shows the fluorescence read out from the streptavidin fluorophore conjugate, and the middle shows the same Western blot imaged with Anti-His₆ fluorophore conjugate. The right most is a gel with the total protein stained by Coomassie. Analysis with A) Cb and B) Hp.