Characterisation of a novel, retaining pseudaminic acid-processing glycosyltransferase enzyme, KpsS1

Nonulosonic acids (NulOs) nine-carbon α-keto acids, the most abundant of which is sialic acid (Neu5Ac). Neu5Ac is found ubiquitously across mammalian and bacterial cell surfaces and is involved, although not exhaustively, in cellular recognition and regulation, intercellular signalling, membrane stabilisation, membrane transport, and cell adhesion. A sub-category of NulOs, known as ‘bacterial NulOs’, are strictly non-mammalian and are found on the surface of several pathogenic bacteria. The first bacterial NulO to be discovered was pseudaminic acid (Pse5Ac7Ac), which has since been found in the cell-surface glycoconjugate structures of multiple pathogenic bacteria. The structural analogy between Pse5Ac7Ac and Neu5Ac confers a fitness advantage to pathogenic bacteria via an immune evasion tactic known as molecular mimicry. Pse5Ac7Ac has been shown to interact with sialic-acid-binding immunoglobulin-like lectin 10 (Siglec-10) receptors on the surface of leukocytes, triggering an anti-inflammatory cascade which culminates in the downregulation of the immune system. As well, knockout of Pse5Ac7Ac biosynthesis genes in gastrointestinal pathogens that ordinarily have pseudaminylated flagella results in aflagellate, immotile mutant phenotypes. Pse5Ac7Ac is therefore an attractive antivirulence drug target. Yet, whilst the Pse5Ac7Ac biosynthetic pathway is well studied, no glycosyltransferases that transfer a Pse5Ac7Ac glycosyl moiety (pseudaminyltransferases, PseTs) had been characterised in vitro prior to this project.
Earlier work putatively assigned an uncharacterised kpss1 gene from Acinetobacter baumannii ACICU as a retaining PseT, and subsequent preliminary in vitro work supported this assignment. This thesis builds upon these observations to describe the purification of KpsS1, and the biochemical assays, bioinformatic and structural studies carried out thereafter which culminated in the establishment of a previously unreported glycosyltransferase family, GT118. As well, upscaled chemoenzymatic syntheses of α-CMP-Pse5Ac7Ac, CMP-3-F-Pse5Ac7Ac, α-Pse5Ac7Ac-2,6-β-D-PNP-Glcp, and α-Pse5Ac7Ac-2,6-β-D-Glc-1,6-β-D-Gal-OMe are reported.