Reaction Mechanism of Glycoside Hydrolase Family 116 Utilizes Perpendicular Protonation

Salila Pengthaisong; Beatriz Piniello; Gideon J Davies; Carme Rovira; James R Ketudat Cairns

doi:10.1021/acscatal.3c00620

Reaction Mechanism of Glycoside Hydrolase Family 116 Utilizes Perpendicular Protonation

ACS Catal. 2023 Apr 14;13(9):5850-5863. doi: 10.1021/acscatal.3c00620. eCollection 2023 May 5.

Authors

Salila Pengthaisong^{1

2}, Beatriz Piniello³, Gideon J Davies⁴, Carme Rovira^{3

5}, James R Ketudat Cairns^{1

2}

Affiliations

¹ School of Chemistry, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand.
² Center for Biomolecular Structure, Function and Application, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand.
³ Departament de Quımica Inorgánica i Orgànica (Secció de Química Orgànica) and Institut de Química Teòrica i Computacional (IQTCUB), Universitat de Barcelona, 08028 Barcelona, Spain.
⁴ Department of Chemistry, University of York, Heslington, York YO10 5DD, U.K.
⁵ Institució Catalana de Recerca i Estudis Avancats (ICREA), 08010 Barcelona, Spain.

Abstract

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 of Thermoanaerobacterium xylanolyticum β-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-type TxGH116 indicate that the substrate binds with the nonreducing glucose residue in an unusual relaxed ⁴C₁ chair at the -1 subsite. Nevertheless, the reaction can still proceed through a ⁴H₃ half-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 a gauche, trans orientation 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.