Abstract
The membrane-associated, processive and retaining glycosyltransferase PglH from Campylobacter jejuni is part of the biosynthetic pathway of the lipid-linked oligosaccharide (LLO) that serves as the glycan donor in bacterial protein N-glycosylation. Using an unknown counting mechanism, PglH catalyzes the transfer of exactly three α1,4 N-acetylgalactosamine (GalNAc) units to the growing LLO precursor, GalNAc-α1,4-GalNAc-α1,3-Bac-α1-PP-undecaprenyl. Here, we present crystal structures of PglH in three distinct states, including a binary complex with UDP-GalNAc and two ternary complexes containing a chemo-enzymatically generated LLO analog and either UDP or synthetic, nonhydrolyzable UDP-CH2-GalNAc. PglH contains an amphipathic helix (“ruler helix”) that has a dual role of facilitating membrane attachment and glycan counting. The ruler helix contains three positively charged side chains that can bind the pyrophosphate group of the LLO substrate and thus limit the addition of GalNAc units to three. These results, combined with molecular dynamics simulations, provide the mechanism of glycan counting by PglH.
Highlights
The membrane-associated, processive and retaining glycosyltransferase PglH from Campylobacter jejuni is part of the biosynthetic pathway of the lipid-linked oligosaccharide (LLO) that serves as the glycan donor in bacterial protein N-glycosylation
To understand the structural basis of such a counting mechanism, we combine structural studies and molecular dynamics simulations with synthetic, chemo-enzymatic, and enzymological studies, and uncover a mechanism by which PglH controls the number of successive glycan transfer reactions using a structural element that attaches the protein to the membrane surface, and contains residues that interact with the pyrophosphate group of the LLO during elongation of the glycan moiety
The chemo-enzymatic generation of triLLO analogs combined with the synthesis of the nonhydrolyzable phosphonate analog of uridine diphosphate (UDP)-GalNAc allowed for the first time the trapping and visualization of a transition-state mimic of a membrane-associated GT-B
Summary
The membrane-associated, processive and retaining glycosyltransferase PglH from Campylobacter jejuni is part of the biosynthetic pathway of the lipid-linked oligosaccharide (LLO) that serves as the glycan donor in bacterial protein N-glycosylation. The ruler helix contains three positively charged side chains that can bind the pyrophosphate group of the LLO substrate and limit the addition of GalNAc units to three. These results, combined with molecular dynamics simulations, provide the mechanism of glycan counting by PglH. To understand the structural basis of such a counting mechanism, we combine structural studies and molecular dynamics simulations with synthetic, chemo-enzymatic, and enzymological studies, and uncover a mechanism by which PglH controls the number of successive glycan transfer reactions using a structural element that attaches the protein to the membrane surface, and contains residues that interact with the pyrophosphate group of the LLO during elongation of the glycan moiety
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