High-resolution structures ofNeotermes koshunensisβ-glucosidase mutants provide insights into the catalytic mechanism and the synthesis of glucoconjugates

Abstract

NkBgl, a β-glucosidase from Neotermes koshunensis, is a β-retaining glycosyl hydrolase family 1 enzyme that cleaves β-glucosidic linkages in disaccharide or glucose-substituted molecules. β-Glucosidases have been widely used in several applications. For example, mutagenesis of the attacking nucleophile in β-glucosidase has been conducted to convert it into a glycosynthase for the synthesis of oligosaccharides. Here, several high-resolution structures of wild-type or mutated NkBgl in complex with different ligand molecules are reported. In the wild-type NkBgl structures it was found that glucose-like glucosidase inhibitors bind to the glycone-binding pocket, allowing the buffer molecule HEPES to remain in the aglycone-binding pocket. In the crystal structures of NkBgl E193A, E193S and E193D mutants Glu193 not only acts as the catalytic acid/base but also plays an important role in controlling substrate entry and product release. Furthermore, in crystal structures of the NkBgl E193D mutant it was found that new glucoconjugates were generated by the conjugation of glucose (hydrolyzed product) and HEPES/EPPS/opipramol (buffer components). Based on the wild-type and E193D-mutant structures of NkBgl, the glucosidic bond of cellobiose or salicin was hydrolyzed and a new bond was subsequently formed between glucose and HEPES/EPPS/opipramol to generate new glucopyranosidic products through the transglycosylation reaction in the NkBgl E193D mutant. This finding highlights an innovative way to further improve β-glucosidases for the enzymatic synthesis of oligosaccharides.

Using the Amino Acid Network to Modulate the Hydrolytic Activity of β-Glycosidases

“…This confirms that solving the Sfβgly crystal structure was fundamental to accurately measure distances among its residue atoms and thus the determination of its structural network. Sfβgly presents the typical (β/α) 8 barrel fold seen for GH1 enzymes (Fig 1B) [3,4,19,31,32]. The recombinant expression of Sfβgly in P .…”

“…The crystal structures of GH1 N . koshunensis β-glycosidase (PDB ID 3AHZ, Figures B-C in S1 Fig) [19] and Brevicoryne brassicae myrosinase (PDB ID 1WCG, Figure D in S1 Fig) [35] also exhibit the same dimeric interface observed for Sfβgly. Moreover, Direct Coupling Analysis (DCA) [29] infers statistical co-evolutionary coupling between Sfβgly residue pairs at the protein surface involved in dimeric contacts: 110–152, 112–150, 111–163, 152–163, 157–163 and 156–211 (Table E in S1 File), supporting that β-glycosidase dimerization is a common, but previously overlooked, feature among insect GH1 enzymes.…”

“…Given the high structural similarity of active sites from Sfβgly and from N . koshunensis β-glycosidase (PDB IDs: 3AI0, complexed to NPβglc, Fig 3D; 3VIK, complexed to cellobiose, S3 Fig) [19], we used their superposition to analyse the fitting of substrates into the Sfβgly active site (Fig 3D; S3 Fig) and correlate structural and mutational data.…”

“…koshunensis [19] and the fungus Trichoderma reesei TrBgl2 [45]. We anticipate as hotspots for future mutagenesis studies the positions corresponding to R189, K201, S247, N249, F251, F334, L350, K366 and M453 (Sfβgly numbering), which are located in the vicinity of substrate aglycone (S3 Fig; Fig 4) and whose mutations enhance the hydrolysis rate of synthetic substrates in Sfβgly, with similar results observed in the TrBgl2 [45] and in Rice Bglu1 [12].…”

“…Collected data were processed in space group P1 using HKL2000 [17]. Initial phases for Sfβgly were obtained by molecular replacement using the program Phaser [18] from the CCP4 package (Collaborative Computational Project, Number 4, 1994) and the structure of the β-glycosidase from Neotermes koshunensis (PDB ID 3VIF) [19] as the search model (46.18% identity with Sfβgly). The unit cell contains six Sfβgly molecules.…”