Reconstitution and optimisation of the biosynthesis of bacterial sugar pseudaminic acid (Pse5Ac7Ac) enables preparative enzymatic synthesis of CMP-Pse5Ac7Ac

Abstract

Pseudaminic acids present on the surface of pathogenic bacteria, including gut pathogens Campylobacter jejuni and Helicobacter pylori, are postulated to play influential roles in the etiology of associated infectious diseases through modulating flagella assembly and recognition of bacteria by the human immune system. Yet they are underexplored compared to other areas of glycoscience, in particular enzymes responsible for the glycosyltransfer of these sugars in bacteria are still to be unambiguously characterised. This can be largely attributed to a lack of access to nucleotide-activated pseudaminic acid glycosyl donors, such as CMP-Pse5Ac7Ac. Herein we reconstitute the biosynthesis of Pse5Ac7Ac in vitro using enzymes from C. jejuni (PseBCHGI) in the process optimising coupled turnover with PseBC using deuterium wash in experiments, and establishing a method for co-factor regeneration in PseH tunover. Furthermore we establish conditions for purification of a soluble CMP-Pse5Ac7Ac synthetase enzyme PseF from Aeromonas caviae and utilise it in combination with the C. jejuni enzymes to achieve practical preparative synthesis of CMP-Pse5Ac7Ac in vitro, facilitating future biological studies.

Figure 1

The nonulosonic acids; Pse5Ac7Ac 1 and Neu5Ac 2.

Scheme 1

H. pylori/C. jejuni biosynthetic pathway from UDP-GlcNAc 4 to CMP-Pse5Ac7Ac 3.

Figure 2

4–20% SDS-PAGE of Ni²⁺-His₆ purified C. jejuni Pse5Ac7Ac biosynthetic enzymes PseBCHGI.

Figure 3

C. jejuni PseB and PseC reaction progression (a) after 45 min of incubation with UDP-GlcNAc 4, and (b) after 6 h of incubation with UDP-GlcNAc 4 showing no significant further reaction progress to the PseC product 6.

Scheme 2

Full C. jejuni PseB and PseC catalysed reactions in water or deuterated buffer.

Figure 4

Negative ESI LC–MS monitoring relative conversion to the PseB and PseC products in deuterated buffer with UDP-GlcNAc 4 (a) after 10 min with equal concentrations of PseB and PseC, (b) after 10 min with a PseB:PseC concentration of 1:5, (c) after 2 h with equal concentrations of PseB and PseC or (d) after 2 h with a PseB:PseC concentration of 1:5.

Scheme 3

In situ regeneration of the acetyltransfer co-factor 5, with acetylthiocholine iodide 16, during the one-pot three enzyme synthesis of the Pse5Ac7Ac biosynthetic intermediate 17.

Figure 5

Negative ESI LC–MS analysis of relative conversion to the PseH product 17 from UDP-GlcNAc 4, investigating the use of acetylthiocholine iodide 16 as a regeneration factor with sub-stoichiometric amounts of Ac-CoA 5 (a) 0 mM Ac-CoA 5 and 20 mM acetylthiocholine iodide 16, (b) 0.15 mM Ac-CoA 5 and 0 mM acetylthiocholine iodide 16, (c) 0.15 mM Ac-CoA 5 and 2 mM acetylthiocholine iodide 16, and (d) 0.15 mM Ac-CoA 5 and 20 mM acetylthiocholine iodide 16.

Scheme 4

“One-pot” chemoenzymatic synthesis of CMP-Pse5Ac7Ac 3 from UDP-GlcNAc 4 using the biosynthetic enzymes under optimised in vitro conditions.