Mechanistic study of CMP-Neu5Ac hydrolysis by α2,3-sialyltransferase from Pasteurella dagmatis.

Bacterial sialyltransferases of the glycosyltransferase family GT-80 exhibit pronounced hydrolase activity toward CMP-activated sialyl donor substrates. Using in situ proton NMR, we show that hydrolysis of CMP-Neu5Ac by Pasteurella dagmatis α2,3-sialyltransferase (PdST) occurs with axial-to-equatorial inversion of the configuration at the anomeric center to release the α-Neu5Ac product. We propose a catalytic reaction through a single displacement-like mechanism where water replaces the sugar substrate as a sialyl group acceptor. PdST variants having His(284) in the active site replaced by Asn, Asp or Tyr showed up to 10(4)-fold reduced activity, but catalyzed CMP-Neu5Ac hydrolysis with analogous inverting stereochemistry. The proposed catalytic role of His(284) in the PdST hydrolase mechanism is to facilitate the departure of the CMP leaving group.