5 years ago

Active-Site His85 of Pasteurella dagmatis Sialyltransferase Facilitates Productive Sialyl Transfer and So Prevents Futile Hydrolysis of CMP-Neu5Ac

Active-Site His85 of Pasteurella dagmatis Sialyltransferase Facilitates Productive Sialyl Transfer and So Prevents Futile Hydrolysis of CMP-Neu5Ac
Manuel Eibinger, Katharina Schmölzer, Bernd Nidetzky
Sialyltransferases of the GT-80 glycosyltransferase family are considered multifunctional because of the array of activities detected. They exhibit glycosyl transfer, trans-sialylation, and hydrolysis activities. How these enzymes utilize their active-site residues in balancing the different enzymatic activities is not well understood. In this study of Pasteurella dagmatis α2,3sialyltransferase, we show that the conserved His85 controls efficiency and selectivity of the sialyl transfer. A His85Asn variant was 200 times less efficient than wild-type for sialylation of lactose, and exhibited relaxed site selectivity to form not only the α2,3- but also the α2,6-sialylated product (21 %). The H85N variant was virtually inactive in trans-sialylation but showed almost the same CMP-Neu5Ac hydrolase activity as wild-type. The competition between sialyl transfer and hydrolysis in the conversion of CMP-Neu5Ac was dependent on the lactose concentration; this was characterized by a kinetic partition ratio of 85 m−1 for the H85N variant, compared to 17 000 m−1 for the wild-type enzyme. His85 promotes the productive sialyl transfer to lactose and so prevents hydrolysis of CMP-Neu5Ac in the reaction. Sialyl transfer versus donor hydrolysis: GT-80 sialyltransferases are also efficient CMP-Neu5Ac hydrolases. His85 is highly conserved in this GT family. We show that His85 controls efficiency and selectivity of sialyl transfer and prevents futile hydrolysis of CMP-Neu5Ac. We present a detailed analysis of the kinetic competition between CMP-Neu5Ac utilization for sialyl transfer and hydrolysis.

Publisher URL: http://onlinelibrary.wiley.com/resolve/doi

DOI: 10.1002/cbic.201700113

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