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Synthesis and application of a highly branched, mechanism-based 2-deoxy-2-fluoro-oligosaccharide inhibitor of endo -xyloglucanases

DOI: 10.1039/C8OB02250J DOI Help

Authors: Namrata Jain (University of British Columbia) , Mohamed A. Attia (University of British Columbia) , Wendy A. Offen (University of York) , Gideon J. Davies (The University of York) , Harry Brumer (University of British Columbia; University of York)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Organic & Biomolecular Chemistry , VOL 5

State: Published (Approved)
Published: October 2018
Diamond Proposal Number(s): 13587

Open Access Open Access

Abstract: Xyloglucan (XyG) is a complex polysaccharide that is ubiquitous and often abundant in the cell walls of terrestrial plants. XyG metabolism is therefore a key component of the global carbon cycle, and hence XyG enzymology is of significant fundamental and applied importance in biomass conversion. To facilitate structure–function analyses of XyG-specific endo-glucanases, we have synthesized a 2′,4′-dinitrophenyl 2-deoxy-2-fluoro-β-glycoside mechanism-based inhibitor based on the highly branched XyG repeating motif XXXG (Xyl3Glc4: ([α-D-Xylp-(1→6)]-β-D-Glcp-(1→4)-[α-D-Xylp-(1→6)]-β-D-Glcp-(1→4)-[α-D-Xylp-(1→6)]-β-D-Glcp-(1→4)-D-Glcp. Key steps in the chemo-enzymatic synthesis included selective enzyme hydrolysis of XyG polysaccharide to produce the core heptasaccharide, per-O-acetylation, α-bromination, reductive glycal formation, electrophilic fluorination, SNAr glycosylation, and Zemplen deprotection. The resulting compound, XXXG(2F)-β-DNP, specifically labelled the active sites of several endo-(xylo)glucanases by accumulation of a covalent glycosyl-enzyme intermediate, as revealed by intact protein mass spectrometry. Crystallography of a complex with a Cellvibrio japonicus Glycoside Hydrolase Family 5 (GH5) endo-xyloglucanase corroborated the covalent nature of the intermediate, and further revealed the anticipated specificity for the catalytic nucleophile of this anomeric-configuration-retaining glycosidase. This specificity complements that of an analogous XXXG N-bromoacetylglycosylamine inhibitor, which labelled the catalytic acid–base sidechain in the same enzyme [Attia, et al., Biotechnol. Biofuels, 2018, 11, 45]. We anticipate that these inhibitors may find continued use in mechanistic analyses of endo-(xylo)glucanases from diverse GH families.

Diamond Keywords: Enzymes

Subject Areas: Chemistry, Biology and Bio-materials

Instruments: I03-Macromolecular Crystallography , I04-1-Macromolecular Crystallography (fixed wavelength)

Added On: 08/11/2018 14:00


Discipline Tags:

Bioenergy Earth Sciences & Environment Physics Climate Change Hard condensed matter - structures Biochemistry Catalysis Chemistry Life Sciences & Biotech

Technical Tags:

Diffraction Macromolecular Crystallography (MX)