Decreasing the sialidase activity of multifunctional Pasteurella multocida α2–3-sialyltransferase 1 (PmST1) by site-directed mutagenesis

Abstract

Pasteurella multocida α2–3-sialyltransferase 1 (PmST1) is a multifunctional enzyme which has α2–6-sialyltransferase, α2–3-sialidase, and α2–3-trans-sialidase activities in addition to its major α2–3-sialyltransferase activity. The presence of the α2–3-sialidase activity of PmST1 complicates its application in enzymatic synthesis of α2–3-linked sialosides as the product formed can be hydrolyzed by the enzyme. Herein we show that the α2–3-sialidase activity of PmST1 can be significantly decreased by protein crystal structure-based site-directed mutagenesis. A PmST1 double mutant E271F/R313Y showed a significantly (6333-fold) decreased sialidase activity without affecting its α2–3-sialyltransferase activity. The double mutant E271F/R313Y, therefore, is a superior enzyme for enzymatic synthesis of α2–3-linked sialosides.