The Retaining Pse5Ac7Ac Pseudaminyltransferase KpsS1 Defines a Previously Unreported glycosyltransferase family (GT118).

Cell surface sugar 5,7-diacetyl pseudaminic acid (Pse5Ac7Ac) is a bacterial analogue of the ubiquitous sialic acid, Neu5Ac, and contributes to the virulence of a number of multidrug resistant bacteria, including ESKAPE pathogens Pseudomonas aeruginosa, and Acinetobacter baumannii. Despite its discovery in the surface glycans of bacteria over thirty years ago, to date no glycosyltransferase enzymes (GTs) dedicated to the synthesis of a pseudaminic acid glycosidic linkage have been unequivocally characterised in vitro. Herein we demonstrate that A. baumannii KpsS1 is a dedicated pseudaminyltransferase enzyme (PseT) which constructs a Pse5Ac7Ac-α(2,6)-Glcp linkage, and proceeds with retention of anomeric configuration. We utilise this PseT activity in tandem with the biosynthetic enzymes required for CMP-Pse5Ac7Ac assembly, in a two-pot, seven enzyme synthesis of an α-linked Pse5Ac7Ac glycoside. Due to its unique activity and protein sequence, we also assign KpsS1 as the prototypical member of a previously unreported GT family (GT118).

Co-factor prosthesis facilitates biosynthesis of azido-pseudaminic acid probes for use as glycosyltransferase reporters.

Truncated thioester N,S-diacetylcysteamine (SNAc) was utilised as a co-factor mimic for PseH, an acetyl-coA dependent aminoglycoside N-acetyltransferase, in the biosynthesis of the bacterial sugar, pseudaminic acid. Additionally, an azido-SNAc analogue was used to smuggle N7-azide functionality into the pseudaminic acid backbone, facilitating its use as a reporter of pseudaminyltransferase activity.

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

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.

Synthetic Approaches for Accessing Pseudaminic Acid (Pse) Bacterial Glycans.

Pseudaminic acids (Pses) are a group of non-mammalian nonulosonic acids (nulOs) that have been shown to be an important virulence factor for a number of pathogenic bacteria, including emerging multidrug-resistant ESKAPE pathogens. Despite their discovery over 30 years ago, relatively little is known about the biological significance of Pse glycans compared with their sialic acid analogues, primarily due to a lack of access to the synthetically challenging Pse architecture. Recently, however, the Pse backbone has been subjected to increasing synthetic exploration by carbohydrate (bio)chemists, and the total synthesis of complex Pse glycans achieved with inspiration from the biosynthesis and subsequent detailed study of chemical glycosylation by using Pse donors. Herein, context is provided for these efforts by summarising recent synthetic approaches pioneered for accessing Pse glycans, which are set to open up this underexplored area of glycoscience to the wider scientific community.