Determinants of undesired α2-6-sialoside formation by PmST1 M144D

Abstract

Sialyltransferases catalyze the regioselective glycosidic bond formation between sialic acid and a glycan acceptor. Pasteurella multocida α2-3-sialyltransferase 1 (PmST1) is a widely used enzyme that has gained widespread use in chemoenzymatic synthesis. In particularly, the PmST1 M144D mutant is routinely employed as an α2-3-sialyltransferase, although low levels of α2-6-sialyltransferase activity have been reported. Here, we discover that for certain acceptors, the formation of the undesired α2-6-sialoside can reach up to 20% of the product. To elucidate the factors that influence this regioselectivity, we systemically examined the effects of (i) sulfation of the acceptor, (ii) chemical nature of the aglycone, (iii) pH, and (iv) extent of reaction completion. Results indicate that sulfation at the 6-position of the GlcNAc or an β-ethyl-NHCBz aglycone are factors that can increase the amount of α2-6 sialoside product. Surprisingly, pH only had a small impact, and the amount of α2-6 sialoside product did not differ over the course of a reaction. These findings provide insights into the enzymatic specificity of PmST1 M144D and inform its optimized use in chemo-enzymatic synthesis of defined sialosides.