Retaining glycoside hydrolases use acid/base catalysis
with an
enzymatic acid/base protonating the glycosidic bond oxygen to facilitate
leaving-group departure alongside attack by a catalytic nucleophile
to form a covalent intermediate. Generally, this acid/base protonates
the oxygen laterally with respect to the sugar ring, which places
the catalytic acid/base and nucleophile carboxylates within about
4.5–6.5 Å of each other. However, in glycoside hydrolase
(GH) family 116, including disease-related human acid β-glucosidase
2 (GBA2), the distance between the catalytic acid/base and the nucleophile
is around 8 Å (PDB:5BVU) and the catalytic acid/base appears to be above the
plane of the pyranose ring, rather than being lateral to that plane,
which could have catalytic consequences. However, no structure of
an enzyme–substrate complex is available for this GH family.
Here, we report the structures ofThermoanaerobacterium
xylanolyticumβ-glucosidase (TxGH116) D593N acid/base mutant in complexes with cellobiose and laminaribiose
and its catalytic mechanism. We confirm that the amide hydrogen bonding
to the glycosidic oxygen is in a perpendicular rather than lateral
orientation. Quantum mechanics/molecular mechanics (QM/MM) simulations
of the glycosylation half-reaction in wild-typeTxGH116 indicate that the substrate binds with the nonreducing glucose
residue in an unusual relaxed4C1chair at the–1subsite. Nevertheless, the
reaction can still proceed through a4H3half-chair transition state, as in classical retaining
β-glucosidases, as the catalytic acid D593 protonates the perpendicular
electron pair. The glucose C6OH is locked in agauche,transorientation with respect to the C5–O5
and C4–C5 bonds to facilitate perpendicular protonation. These
data imply a unique protonation trajectory in Clan-O glycoside hydrolases,
which has strong implications for the design of inhibitors specific
to either lateral protonators, such as human GBA1, or perpendicular
protonators, such as human GBA2.