Fragment-derived modulators of an industrial β-glucosidase

TY - JOUR

T1 - Fragment-derived modulators of an industrial β-glucosidase

AU - Makraki, Eleni

AU - Darby, John F.

AU - Carneiro, Marta G.

AU - Firth, James D.

AU - Heyam, Alex

AU - Ab, Eiso

AU - O'Brien, Peter

AU - Siegal, Gregg

AU - Hubbard, Roderick E.

N1 - © 2020 The Author(s)

PY - 2020/11/26

Y1 - 2020/11/26

N2 - 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.

AB - 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.

KW - TrBgl2

KW - glycoside hydrolase

KW - NMR spectroscopy

KW - protein–ligand docking

KW - small molecule activators

UR - http://www.scopus.com/inward/record.url?scp=85096947075&partnerID=8YFLogxK

U2 - 10.1042/BCJ20200507

DO - 10.1042/BCJ20200507

M3 - Article

C2 - 33111951

AN - SCOPUS:85096947075

SN - 0264-6021

VL - 477

SP - 4383

EP - 4395

JO - The Biochemical journal

JF - The Biochemical journal

IS - 22

ER -