Dataset Information

Expression and crystallization of a bacterial glycoside hydrolase family 116 ?-glucosidase from Thermoanaerobacterium xylanolyticum.

ABSTRACT: The Thermoanaerobacterium xylanolyticum gene product TxGH116, a glycoside hydrolase family 116 protein of 806 amino-acid residues sharing 37% amino-acid sequence identity over 783 residues with human glucosylceramidase 2 (GBA2), was expressed in Escherichia coli. Purification by heating, immobilized metal-affinity and size-exclusion chromatography produced >90% pure TxGH116 protein with an apparent molecular mass of 90?kDa on SDS-PAGE. The purified TxGH116 enzyme hydrolyzed the p-nitrophenyl (pNP) glycosides pNP-?-D-glucoside, pNP-?-D-galactoside and pNP-N-acetyl-?-D-glucopyranoside, as well as cellobiose and cellotriose. The TxGH116 protein was crystallized using a precipitant consisting of 0.6?M sodium citrate tribasic, 0.1?M Tris-HCl pH 7.0 by vapour diffusion with micro-seeding to form crystals with maximum dimensions of 120×25×5?µm. The TxGH116 crystals diffracted X-rays to 3.15?Å resolution and belonged to the monoclinic space group P2(1). Structure solution will allow a structural explanation of the effects of human GBA2 mutations.

SUBMITTER: Sansenya S

PROVIDER: S-EPMC4304745 | biostudies-literature | 2015 Jan

REPOSITORIES: biostudies-literature

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Expression and crystallization of a bacterial glycoside hydrolase family 116 β-glucosidase from Thermoanaerobacterium xylanolyticum.

Sansenya Sompong S Mutoh Risa R Charoenwattanasatien Ratana R Kurisu Genji G Ketudat Cairns James R JR

Acta crystallographica. Section F, Structural biology communications 20150101 Pt 1

The Thermoanaerobacterium xylanolyticum gene product TxGH116, a glycoside hydrolase family 116 protein of 806 amino-acid residues sharing 37% amino-acid sequence identity over 783 residues with human glucosylceramidase 2 (GBA2), was expressed in Escherichia coli. Purification by heating, immobilized metal-affinity and size-exclusion chromatography produced >90% pure TxGH116 protein with an apparent molecular mass of 90 kDa on SDS-PAGE. The purified TxGH116 enzyme hydrolyzed the p-nitrophenyl (pN ...[more]

PMID: 25615966

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Project description: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.

Dataset Information

Expression and crystallization of a bacterial glycoside hydrolase family 116 ?-glucosidase from Thermoanaerobacterium xylanolyticum.

Publications

Expression and crystallization of a bacterial glycoside hydrolase family 116 β-glucosidase from Thermoanaerobacterium xylanolyticum.

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