Molecular Mechanism of Double-Displacement Retaining β‑Kdo Glycosyltransferase WbbB

Glycosyltransferases (GTs) are pivotal enzymes involved
in glycosidic
bond synthesis, which can lead to either retention or inversion of
the glycosyl moiety’s anomeric configuration. However, the
catalytic mechanism for retaining GTs remains a subject of controversy.
In this study, we employ MD and QM/MM metadynamics to investigate
the double-displacement catalytic mechanism of the retaining β-Kdo
transferase WbbB. Our findings demonstrate that the nucleophile Asp232
initiates the reaction by attacking the sugar ring containing a carboxylate
at the anomeric position, forming a covalent adduct. Subsequently,
the adduct undergoes a rotational rearrangement, ensuring proper orientation
of the anomeric carbon for the acceptor substrate. In the second step,
Glu158 acts as the catalytic base to abstract the proton of the acceptor
substrate to complete the transglycosylation reaction. Notably, His265
does not function as the anticipated catalytic acid; instead, it stabilizes
the phosphate group through H-bonding interactions. Our simulations
support the double-displacement mechanism implicated from the crystallographic
studies of WbbB. This mechanism deviates from the common SNi-type and retaining glycoside hydrolase mechanisms,
thereby expanding our understanding of GT catalytic mechanisms.