Unexpected reactivity and mechanism of carboxamide activation in bacterial N-linked protein glycosylation

Abstract

The initial glycan transfer in asparagine-linked protein glycosylation is catalysed by the integral membrane enzyme oligosaccharyltransferase (OST). Here we study the mechanism of the bacterial PglB protein, a single-subunit OST, using chemically synthesized acceptor peptide analogues. We find that PglB can glycosylate not only asparagine but also glutamine, homoserine and the hydroxamate Asp(NHOH), although at much lower rates. In contrast, N-methylated asparagine or 2,4-diaminobutanoic acid (Dab) are not glycosylated. We find that of the various peptide analogues, only asparagine-or Dab-containing peptides bind tightly to PglB. Glycopeptide products are unable to bind, providing the driving force of product release. We find no suitably positioned residues near the active site of PglB that can activate the acceptor asparagine by deprotonation, making a general base mechanism unlikely and leaving carboxamide twisting as the most likely mechanistic proposal for asparagine activation.

Structure and mechanism of an active lipid-linked oligosaccharide flippase

“…It is important to note that reductions of the in vivo output of the biosynthetic glycosylation pathway as a consequence of a PglK mutation may not directly correlate with the reduction of in vitro flipping rates because the rate-limiting step of the in vivo process is unknown. A similar phenomenon was previously observed for mutants of PglB that exhibited reductions of in vitro glycosylation rates by two orders of magnitude or more, but showing only marginally lower in vivo output 21,26,30,31 . We speculated that the hydrophobic grooves formed near the EH (Fig.…”

“…The reaction was stopped by heat shock (90 uC for 10 min). The products were then analysed by in vitro glycosylation of a fluorescently labelled substrate peptide (DQNAT sequon) catalysed by PglB 30,31 . In brief, the reaction products were incubated in a mixture containing 10 mM MES, pH 6.5, 100 mM NaCl, 10 mM MnCl 2 , 3% glycerol (v/v), 1% Triton X-100 (w/v), 0.5 mM fluorescently labelled acceptor peptide and 1 mM purified PglB.…”

“…9a). The conversion of tLLO to hexa-saccharide LLO was confirmed by transferring the resulting oligosaccharides to fluorescently labelled acceptor peptides using purified oligosaccharyltransferase PglB, followed by SDS-PAGE analysis 26,30,31 . PglK-catalysed in vitro tLLO flipping was strictly dependent on ATP hydrolysis.…”

A Catalytically Essential Motif in External Loop 5 of the Bacterial Oligosaccharyltransferase PglB

“…Through a combination of systematic mutagenesis and in vivo activity analysis, we now identified a previously unrecognized motif that proved essential for catalytic activity. Unexpectedly, mutations in the newly found Tyr-plug had stronger effects on PglB activity than many of the previously described mutations at the active site, even of catalytically essential residues (13,14). However, replacing the Tyrplug sequence did not affect substrate peptide binding.…”

“…Recent functional studies have provided a quantitative basis for sequon recognition and binding by PglB; the ϩ2 sequon Ser/Thr of acceptor substrates is recognized by a binding pocket provided by the WWD motif (a diagnostic motif among Stt3 homologs) and a neighboring Ile residue (Ile 572 ), suggesting that the ϩ2 Ser/Thr defines the specificity of N-linked glycosylation sites (13). The C. lari PglB enzyme was also useful for studying the mechanism of activation of the acceptor Asn side chain of sequon (14).…”