Fragment-derived modulators of an industrial β-glucosidase

Eleni Makraki, John F. Darby, Marta G. Carneiro, James D. Firth, Alex Heyam, Eiso Ab, Peter O'Brien, Gregg Siegal, Roderick E. Hubbard

Research output: Contribution to journalArticlepeer-review


A fragment screen of a library of 560 commercially available fragments using a kinetic assay identified a small molecule that increased the activity of the fungal glycoside hydrolase TrBgl2. An analogue by catalogue approach and detailed kinetic analysis identified improved compounds that behaved as nonessential activators with up to a 2-fold increase in maximum activation. The compounds did not activate the related bacterial glycoside hydrolase CcBglA demonstrating specificity. Interestingly, an analogue of the initial fragment inhibits both TrBgl2 and CcBglA, apparently through a mixed-model mechanism. Although it was not possible to determine crystal structures of activator binding to 55 kDa TrBgl2, solution NMR experiments demonstrated a specific binding site for the activator. A partial assignment of the NMR spectrum gave the identity of the amino acids at this site, allowing a model for TrBgl2 activation to be built. The activator binds at the entrance of the substrate-binding site, generating a productive conformation for the enzyme-substrate complex.

Original languageEnglish
Pages (from-to)4383-4395
Number of pages13
JournalThe Biochemical journal
Issue number22
Publication statusPublished - 26 Nov 2020

Bibliographical note

© 2020 The Author(s)


  • TrBgl2
  • glycoside hydrolase
  • NMR spectroscopy
  • protein–ligand docking
  • small molecule activators

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