TY - JOUR
T1 - Mechanistic study of CMP-Neu5Ac hydrolysis by α2,3-sialyltransferase from Pasteurella dagmatis
AU - Schmoelzer, Katharina
AU - Luley, Christiane
AU - Czabany, Tibor
AU - Ribitsch, Doris
AU - Schwab, Helmut
AU - Weber, Hansjörg
AU - Nidetzky, Bernd
PY - 2014
Y1 - 2014
N2 - 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 His284 in the active site replaced by Asn, Asp or Tyr showed up to 104-fold reduced activity, but catalyzed CMP-Neu5Ac hydrolysis with analogous inverting stereochemistry. The proposed catalytic role of His284 in the PdST hydrolase mechanism is to facilitate the departure of the CMP leaving group.
AB - 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 His284 in the active site replaced by Asn, Asp or Tyr showed up to 104-fold reduced activity, but catalyzed CMP-Neu5Ac hydrolysis with analogous inverting stereochemistry. The proposed catalytic role of His284 in the PdST hydrolase mechanism is to facilitate the departure of the CMP leaving group.
U2 - 10.1016/j.febslet.2014.05.053
DO - 10.1016/j.febslet.2014.05.053
M3 - Article
SN - 1873-3468
VL - 588
SP - 2978
EP - 2984
JO - FEBS Letters
JF - FEBS Letters
IS - 17
ER -