Decreasing the sialidase activity of multifunctional Pasteurella multocida alpha 2-3-sialyltransferase 1 (PmST1) by site-directed mutagenesis

Pasteurella multocida alpha 2-3-sialyltransferase 1 (PmST1) is a multifunctional enzyme which has alpha 2-6-sialyltransferase, alpha 2-3-sialidase, and alpha 2-3-trans-sialidase activities in addition to its major alpha 2-3-sialyltransferase activity. The presence of the alpha 2-3-sialidase activity of PmST1 complicates its application in enzymatic synthesis of alpha 2-3-linked sialosides as the product formed can be hydrolyzed by the enzyme. Herein we show that the alpha 2-3-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 alpha 2-3-sialyltransferase activity. The double mutant E271F/R313Y, therefore, is a superior enzyme for enzymatic synthesis of alpha 2-3-linked sialosides.