Investigation on the substrate specificity and -substitution tolerance of PseF in catalytic transformation of pseudaminic acids to CMP-Pse derivatives.

Pseudaminic acid (Pse) belongs to a class of bacterial non-2-ulosonic acids, and has been implicated in bacterial infection and immune evasion. Various Pse structures with diverse -substitutions have been identified in pathogenic bacterial strains like , , and . In this study, we successfully synthesized three new Pse species, including Pse5Ac7Fo, Pse5Ac7(3Hb) and Pse7Fo5(3Hb) using chemical methods. Furthermore, we investigated the substrate specificity of cytidine 5'-monophosphate (CMP)-Pse synthetase (PseF), resulting in the production of -modified CMP-Pse derivatives (CMP-Pses). It was found that PseF was promiscuous with the Pse substrate and could tolerate different modifications at the two nitrogen atoms. This study provides valuable insights into the incorporation of variable -substitutions in the Pse biosynthetic pathway.