A single amino acid toggles Escherichia coli polysialyltransferases between mono- and bifunctionality.

The enteropathogenic Escherichia coli K92 synthesizes a unique capsule consisting of polysialic acid (polySia) with alternating α2,8- and α2,9-linkages. The fact that a single enzyme is responsible for the synthesis of these alternating regioisomeric linkages raises questions as to how this controlled bifunctionality is achieved mechanistically. Aiming to identify the sequence elements responsible for dual regiospecificity, we have utilized a high-throughput polysialyltransferase (polyST) activity screen to explore the relevant sequence space between this enzyme and its close monofunctional homolog from E. coli K1. The linkage specificity of selected mutants was subsequently confirmed using a polySia permethylation linkage analysis technique. We have identified a single amino acid exchange at residue 52 that toggles these enzymes between mono and dual regiospecificity. The results have implications for the mechanism by which the E. coli K92 polyST achieves bifunctional elongation.