Probing Monotopic Phosphoglycosyl Transferases from Complex Cellular Milieu.

Area of Science:

• Biochemistry
• Molecular Biology
• Microbiology

Background:

• Monotopic phosphoglycosyl transferase enzymes (monoPGTs) are vital for prokaryotic glycoconjugate assembly, impacting bacterial survival and proliferation.
• The monoPGT superfamily is large and diverse, but the specific biochemical functions of most members in glycoconjugate biosynthesis remain largely unknown.
• Understanding monoPGT activity is crucial for deciphering bacterial cellular processes and identifying potential therapeutic targets.

Purpose of the Study:

• To develop and apply novel tools for studying the activity and regulation of the monoPGT superfamily within the cellular proteome.
• To investigate the feasibility of using activity-based protein profiling (ABPP) for analyzing membrane-associated monoPGTs.
• To characterize the active site labeling of representative monoPGTs using specific chemical probes.

Main Methods:

• Implementation of activity-based protein profiling (ABPP) probes designed for protein-centric analysis.
• Utilized membrane protein-compatible probes to ensure effective labeling of cell-associated enzymes.
• Employed gel-based readouts for straightforward detection and analysis of enzyme activity following probe labeling.

Main Results:

• Demonstrated robust, covalent labeling of the active sites of various representative monoPGTs.
• Successfully applied ABPP probes to analyze monoPGTs within bacterial cell membrane fractions.
• The 3-phenyl-2H-azirine probes effectively targeted and labeled key monoPGT enzymes.

Conclusions:

• Activity-based protein profiling provides a powerful approach for studying the monoPGT enzyme superfamily.
• This method enables the investigation of monoPGT activity and regulation directly within the cellular proteome.
• The findings lay the groundwork for future research into the diverse roles of monoPGTs in bacterial biology.