posted on 2021-03-24, 17:20authored byCaroline
S. Pereira, Rodrigo L. Silveira, Munir S. Skaf
Glycoside hydrolases
(GH) cleave carbohydrate glycosidic bonds
and play pivotal roles in living organisms and in many industrial
processes. Unlike acid-catalyzed hydrolysis of carbohydrates in solution,
which can occur either via cyclic or acyclic oxocarbenium-like transition
states, it is widely accepted that GH-catalyzed hydrolysis proceeds
via a general acid mechanism involving a cyclic oxocarbenium-like
transition state with protonation of the glycosidic oxygen. The GH45
subfamily C inverting endoglucanase from Phanerochaete chrysosporium (PcCel45A) defies the classical inverting mechanism as its crystal
structure conspicuously lacks a general Asp or Glu base residue. Instead,
PcCel45A has an Asn residue, a notoriously weak base in solution,
as one of its catalytic residues at position 92. Moreover, unlike
other inverting GHs, the relative position of the catalytic residues
in PcCel45A impairs the proton abstraction from the nucleophilic water
that attacks the anomeric carbon, a key step in the classical mechanism.
Here, we investigate the viability of an endocyclic mechanism for
PcCel45A using hybrid quantum mechanics/molecular mechanics (QM/MM)
simulations, with the QM region treated with the self-consistent-charge
density-functional tight-binding level of theory. In this mechanism,
an acyclic oxocarbenium-like transition state is stabilized leading
to the opening of the glucopyranose ring and formation of an unstable
acyclic hemiacetal that can be readily decomposed into hydrolysis
product. In silico characterization of the Michaelis
complex shows that PcCel45A significantly restrains the sugar ring
to the 4C1 chair conformation at the −1
subsite of the substrate binding cleft, in contrast to the classical
exocyclic mechanism in which ring puckering is critical. We also show
that PcCel45A provides an environment where the catalytic Asn92 residue
in its standard amide form participates in a cooperative hydrogen
bond network resulting in its increased nucleophilicity due to an
increased negative charge on the oxygen atom. Our results for PcCel45A
suggest that carbohydrate hydrolysis catalyzed by GHs may take an
alternative route from the classical mechanism.