A novel β-xylosidase structure fromGeobacillus thermoglucosidasius: the first crystal structure of a glycoside hydrolase family GH52 enzyme reveals unpredicted similarity to other glycoside hydrolase folds

Abstract

Geobacillus thermoglucosidasius is a thermophilic bacterium that is able to ferment both C6 and C5 sugars to produce ethanol. During growth on hemicellulose biomass, an intracellular β-xylosidase catalyses the hydrolysis of xylo-oligosaccharides to the monosaccharide xylose, which can then enter the pathways of central metabolism. The gene encoding a G. thermoglucosidasius β-xylosidase belonging to CAZy glycoside hydrolase family GH52 has been cloned and expressed in Escherichia coli. The recombinant enzyme has been characterized and a high-resolution (1.7 Å) crystal structure has been determined, resulting in the first reported structure of a GH52 family member. A lower resolution (2.6 Å) structure of the enzyme-substrate complex shows the positioning of the xylobiose substrate to be consistent with the proposed retaining mechanism of the family; additionally, the deep cleft of the active-site pocket, plus the proximity of the neighbouring subunit, afford an explanation for the lack of catalytic activity towards the polymer xylan. Whilst the fold of the G. thermoglucosidasius β-xylosidase is completely different from xylosidases in other CAZy families, the enzyme surprisingly shares structural similarities with other glycoside hydrolases, despite having no more than 13% sequence identity.

Bacterial β-Glucosidase Reveals the Structural and Functional Basis of Genetic Defects in Human Glucocerebrosidase 2 (GBA2)

“…Tx GH116 shows no structural similarity to other retaining β-glucosidases, which fall into families including GH1, GH2, GH3, GH5, and GH30 ( SI, Figure S2 ). Comparison to structures in the PDB database with PDBeFold 23 found that the Tx GH116 structure is most similar to the family GH52 β-xylosidase from Geobacillus thermoglucosidasius ( Gt GH52, PDB ID: 4C1O, 4C1P), 24 which upon global superposition yields an RMSD of 3.2 Å over 560 matched Cα atoms (PDBeFold Z score 8.3; SI, Figure S3A ). However, the sequence similarity of GH52 and GH116 is negligible (GH52 enzymes are not detected using PSI-BLAST with the Tx GH116 sequence as a search query), GH52 lacks the N-terminal loop that contributes to the entrance to the active site in GH116, and different numbers of strands are found in the major β-sheets of the N-terminal domain (see SI ).…”

P13, the EMBL macromolecular crystallography beamline at the low-emittance PETRA III ring for high- and low-energy phasing with variable beam focusing

“…Highlights from the available publications include the elucidation of the structural basis of the proinflammatory signalling complex mediated by TSLP (Verstraete et al, 2014); the crystal structure of the eukaryotic translation initiation factor eIF5B (399-852) from Saccharomyces cerevisiae (Kuhle & Ficner, 2014); the structure and thermodynamics of inhibition of Sporosarcina pasteurii urease (Benini et al, 2014); crystallographic structures of an active Spiegelmer, NOX-D20, bound to its physiological targets, mouse C5a and C5a-desArg (Yatime et al, 2015). Full MAD and Se-SAD capability are implemented as shown by the crystal structure of Geobacillus thermoglucosidasius GH family 52 xylosidase (Espina et al, 2014) solved with Se-SAD data, or by the in-house structure solution of glucose isomerase with a fluorescence scan around the Pb L III edge at 13050 eV (data not shown).…”

β-Xylosidases: Structural Diversity, Catalytic Mechanism, and Inhibition by Monosaccharides

“…The GH52 β-xylosidases are very similar to each other with amino acid sequence identities of around 41%–90%. In this family, crystal structures are available for β-xylosidases from Parageobacillus thermoglucosidasius TM242 (GT2_24_00240; PDB 4C1O; Figure 2i) [73] and G. stearothermophilus T-6 (Xyn52B2; PDB 4RHH) (Dann et al, unpublished work). With a sequence identity of 86%, the two proteins fold into almost the same structures; they display two distinct domains, an N-terminal β-sandwich domain and a C-terminal (α/α) 6 -barrel domain.…”

“…With a sequence identity of 86%, the two proteins fold into almost the same structures; they display two distinct domains, an N-terminal β-sandwich domain and a C-terminal (α/α) 6 -barrel domain. The catalytic residues of the GH52 enzymes, which are Glu-357 and Asp-517 in GT2_24_00240 [73], are located in the (α/α) 6 -barrel domain. Protein homology modeling based on the structure of GT2_24_00240 suggested that the domains are conserved among the GH52 β-xylosidases, except for the C-terminal domain of GsXylA.…”