Rational engineering of a pseudaminic acid synthase enzyme enables access to a 3-fluoro sugar with potential as a metabolic inhibitor of motility in bacterial pathogens

We report the rational engineering of a pseudaminic acid synthase (PseI) which enables the first synthesis of a 3-fluorinated pseudaminic acid sugar (3-(eq)-F-Pse5Ac7Ac), potentially establishing a new class of metabolic inhibitors targeting bacterial glycosylation. Pseudaminic acids are ⍺-keto acid sugars essential for O-glycosylation of flagellin in pathogens such as Campylobacter jejuni, where they are critical for motility and virulence. By introducing rational mutations in the PseI active site, we reversed the co-factor preference of the enzyme and show enhanced turnover with unnatural 3-fluoro-phosphoenolpyruvate, facilitating a scalable chemoenzymatic synthesis of the fluorinated sugar. Subsequent treatment of C. jejuni with 3-(eq)-F-Pse5Ac7Ac resulted in a significant, time-dependent reduction in motility, and in vitro studies demonstrated bacterial CMP- pseudaminic acid synthetase enzymes (PseF) can process the fluoro sugar to afford CMP-3-(eq)-F- Pse5Ac7Ac, potentially implicating the fluorinated glycosyltransferase donor as an anti-motilin in vivo. This study demonstrates, for the first time, that fluorinated pseudaminic acids can impair bacterial motility, paving the way for anti-virulence strategies in pathogenic bacteria. This anti-motilin approach offers a promising alternative to traditional antibiotics, addressing the urgent need for novel strategies to combat antimicrobial resistance, and could be extended to other bacterial ⍺-keto acid sugars.