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Mechanistic study of CMP‐Neu5Ac hydrolysis by α2,3‐sialyltransferase from Pasteurella dagmatis
Author(s) -
Schmölzer Katharina,
Luley-Goedl Christiane,
Czabany Tibor,
Ribitsch Doris,
Schwab Helmut,
Weber Hansjörg,
Nidetzky Bernd
Publication year - 2014
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/j.febslet.2014.05.053
Subject(s) - sialyltransferase , chemistry , anomer , hydrolase , hydrolysis , stereochemistry , catalysis , glycosyltransferase , mutarotation , active site , enzyme , biochemistry
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.