Several bacterial sialyltransferases have been reported to be multifunctional also catalysing sialidase and trans-sialidase reactions. In this study, we examined the trans-sialylation efficacy and regioselectivity of mutants of the multifunctional Pasteurella multocida sialyltransferase (PmST) for catalysing the synthesis of 3'- and 6'-sialyllactose using casein glycomacropeptide as sialyl-donor and lactose as acceptor. The mutation P34H led to a 980-fold increase in alpha-2,6-sialyltransferase activity (with cytidine-5'-monophospho-N-acetylneuraminic acid as donor), while its alpha-2,3-sialyltransferase activity was abolished. Histidine in this position is conserved in alpha-2,6-sialyltransferases and has been suggested, and recently confirmed, to be the determinant for strict regiospecificity in the sialyltransferase reaction. Our data verified this theorem. In trans-sialidase reactions, the P34H mutant displayed a distinct preference for 6'-sialyllactose synthesis but low levels of 3'-sialyllactose were also produced. The sialyllactose yield was however lower than when using PmSTWT under optimal conditions for 6'-sialyllactose formation. The discrepancy in regiospecificity between the two reactions could indicate subtle differences in the substrate binding site in the two reactions. In contrast, the two mutations E271F and R313Y led to preferential synthesis of 3'-sialyllactose over 6'-sialyllactose and the double mutant (PmSTE271F/R313Y) exhibited the highest alpha-2,3-regioselectivity via reduced sialidase and alpha-2,6-trans-sialidase activity. The double mutant PmSTE271F/R313Y thus showed the highest alpha-2,3-regioselectivity and constitutes an interesting enzyme for regioselective synthesis of alpha-2,3-sialylated glycans. This study has expanded the understanding of the structure-function relationship of multifunctional, bacterial sialyltransferases and provided new enzymes for regioselective glycan sialylation. (C) 2015 Elsevier Inc. All rights reserved.