Retaining Glycosyltransferase Mechanism Studied by QM/MM Methods: Lipopolysaccharyl-α-1,4-galactosyltransferase C Transfers α-Galactose via an Oxocarbenium Ion-like Transition State

Glycosyltransferases (GTs) catalyze the highly specific
biosynthesis
of glycosidic bonds and, as such, are important both as drug targets
and for biotechnological purposes. Despite their broad interest, fundamental
questions about their reaction mechanism remain to be answered, especially
for those GTs that transfer the sugar with net retention of the configuration
at the anomeric carbon (retaining glycosyltransferases, ret-GTs).
In the present work, we focus on the reaction catalyzed by lipopolysaccharyl-α-1,4-galactosyltransferase
C (LgtC) from Neisseria meningitides. We study and
compare the different proposed mechanisms (SNi, SNi-like, and double displacement mechanism via a covalent glycosyl-enzyme
intermediate, CGE) by using density functional theory (DFT) and quantum
mechanics/molecular mechanics (QM/MM) calculations on the full enzyme.
We characterize a dissociative single-displacement (SNi)
mechanism consistent with the experimental data, in which the acceptor
substrate attacks on the side of the UDP leaving group that acts as
a catalytic base. We identify several key interactions that help this
front-side attack by stabilizing the transition state. Among them,
Gln189, the putative nucleophile in a double displacement mechanism,
is shown to favor the charge development at the anomeric center by
about 2 kcal/mol, compatible with experimental mutagenesis data. We
predict that using 3-deoxylactose as acceptor would result in a reduction
of kcat to 0.6–3% of that for the
unmodified substrates. The reactions of the Q189A and Q189E mutants
have also been investigated. For Q189E, there is a change in mechanism
since a CGE can be formed which, however, is not able to evolve to
products. The current findings are discussed in the light of the available
experimental data and compared with those for other ret-GTs.