8I5U

Crystal structure of TxGH116 D593N acid/base mutant from Thermoanaerobacterium xylanolyticum with laminaribiose

Summary for 8I5U

• Entry DOI: 10.2210/pdb8i5u/pdb
• Related: 5BVU 5BX2 5BX3 5BX4 5BX5
• Related PRD ID: PRD_900024
• Descriptor: beta-glucosidase, beta-D-glucopyranose-(1-3)-beta-D-glucopyranose, GLYCEROL, ... (6 entities in total)
• Functional Keywords: hydrolase
• Biological source: Thermoanaerobacterium xylanolyticum LX-11
• Total number of polymer chains: 1
• Total formula weight: 93773.21

Authors

Pengthaisong, S.,Ketudat Cairns, J.R. (deposition date: 2023-01-26, release date: 2023-05-03, Last modification date: 2023-11-29)

Primary citation

Pengthaisong, S.,Piniello, B.,Davies, G.J.,Rovira, C.,Ketudat Cairns, J.R.
Reaction Mechanism of Glycoside Hydrolase Family 116 Utilizes Perpendicular Protonation.
Acs Catalysis, 13:5850-5863, 2023

PubMed 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 β-glucosidase (GH116) 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 GH116 indicate that the substrate binds with the nonreducing glucose residue in an unusual relaxed chair at the subsite. Nevertheless, the reaction can still proceed through a 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 , 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.

PubMed: 37180965
DOI: 10.1021/acscatal.3c00620

Experimental method

X-RAY DIFFRACTION (1.4 Å)