Chemoenzymatic synthesis of polyprenyl phosphates

Abstract

Polyprenyl phosphates, including undecaprenyl phosphate and dolichyl phosphate, are essential intermediates in several important biochemical pathways including N-linked protein glycosylation in eukaryotes and prokaryotes and prokaryotic cell wall biosynthesis. Herein, we describe the evaluation of three potential undecaprenol kinases as agents for the chemoenzymatic synthesis of polyprenyl phosphates. Target enzymes were expressed in crude cell envelope fractions and quantified via the use of luminescent lanthanide-binding tags (LBTs). The Streptococcus mutans diacylglycerol kinase (DGK) was shown to be a very useful agent for polyprenol phosphorylation using ATP as the phosphoryl transfer agent. In addition, the S. mutans DGK can be coupled with two Campylobacter jejuni glycosyltransferases involved in N-linked glycosylation to efficiently biosynthesize the undecaprenyl pyrophosphate-linked disaccharide needed for studies of PglB, the C. jejuni oligosaccharyl transferase.

Figure 1

The C. jejuni N-linked glycosylation pathway. Glycosyltransferases are responsible for the transfer of one or more sugars from the UDP-activated donors (enzyme b–f). Enzymes involved in the pathway: (a) kinase; (b) PglC; (c) PglA; (d) PglJ; (e) PglH; (f) PglI; (g) PglB, the oligosaccharyltransferase; (h) Und-PP phosphatase; Und, undecaprenol; Und-P, undecaprenyl phosphate; Und-PP, undecaprenyl pyrophosphate.

Figure 2

Undecaprenol (1) and dolichol (3) can be phosphorylated to form undecaprenyl phosphate (2) and dolichyl phosphate (4), respectively. This reaction can be done synthetically or chemoenzymatically by the action of a kinase.

Figure 3

An enzymatic reaction scheme showing the synthesis of undecaprenyl pyrophosphate-linked disaccharide (7).

Figure 3

An enzymatic reaction scheme showing the synthesis of undecaprenyl pyrophosphate-linked disaccharide (7).

Figure 4

Time course of assay in which the kinase candidates are coupled to PglC and PglA. Dotted line, S. mutans DGK; dashed line, C. jejuni DGK; solid line, blank cell envelope fraction.

Figure 5

A PglB assay scheme showing transfer of the radiolabeled disaccharide from the isoprenyl pyrophosphate carrier onto a peptide.

Figure 5

A PglB assay scheme showing transfer of the radiolabeled disaccharide from the isoprenyl pyrophosphate carrier onto a peptide.

Figure 6

A time course of a PglB reaction in which radiolabeled undecaprenyl pyrophosphate disaccharide is used as a substrate.