6HAA

Structure of a covalent complex of endo-Xyloglucanase from Cellvibrio japonicus after reacting with XXXG(2F)-beta-DNP

Summary for 6HAA

• Entry DOI: 10.2210/pdb6haa/pdb
• Descriptor: Cellulase, putative, cel5D, beta-D-glucopyranose-(1-4)-[alpha-D-xylopyranose-(1-6)]beta-D-glucopyranose-(1-4)-[alpha-D-xylopyranose-(1-6)]beta-D-glucopyranose-(1-4)-2-deoxy-2-fluoro-alpha-D-glucopyranose, alpha-D-xylopyranose-(1-6)-beta-D-glucopyranose-(1-4)-[alpha-D-xylopyranose-(1-6)]beta-D-glucopyranose-(1-4)-[alpha-D-xylopyranose-(1-6)]beta-D-glucopyranose-(1-4)-2-deoxy-2-fluoro-alpha-D-glucopyranose, ... (9 entities in total)
• Functional Keywords: glycoside hydrolase, xyloglucan, covalent inhibitor, hydrolase
• Biological source: Cellvibrio japonicus (Pseudomonas fluorescens subsp. cellulosa)
• Total number of polymer chains: 2
• Total formula weight: 92071.52

Authors

Offen, W.,Davies, G.J. (deposition date: 2018-08-07, release date: 2018-10-03, Last modification date: 2024-11-13)

Primary citation

Jain, N.,Attia, M.A.,Offen, W.A.,Davies, G.J.,Brumer, H.
Synthesis and application of a highly branched, mechanism-based 2-deoxy-2-fluoro-oligosaccharide inhibitor of endo-xyloglucanases.
Org. Biomol. Chem., 16:8732-8741, 2018

PubMed 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.

PubMed: 30387796
DOI: 10.1039/c8ob02250j

Experimental method

X-RAY DIFFRACTION (1.7 Å)