Revealing the mechanism for covalent inhibition of glycoside hydrolases by carbasugars at an atomic level

Weiwu Ren,Tracey M Gloster,Oluwafemi Akintola,Michael Meanwell,Jason Draper,Robert Pengelly,Marco Farren-Dai,Verena Oehler,Katarzyna Świderek,Saswati Chakladar,Saeideh Shamsi Kazem Abadi,Vicent Moliner,Robert Britton,Andrew J Bennet

doi:10.1038/s41467-018-05702-7

Weiwu Ren, Tracey M Gloster + Show 12 more

Open Access

https://doi.org/10.1038/s41467-018-05702-7

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Abstract

Mechanism-based glycoside hydrolase inhibitors are carbohydrate analogs that mimic the natural substrate’s structure. Their covalent bond formation with the glycoside hydrolase makes these compounds excellent tools for chemical biology and potential drug candidates. Here we report the synthesis of cyclohexene-based α-galactopyranoside mimics and the kinetic and structural characterization of their inhibitory activity toward an α-galactosidase from Thermotoga maritima (TmGalA). By solving the structures of several enzyme-bound species during mechanism-based covalent inhibition of TmGalA, we show that the Michaelis complexes for intact inhibitor and product have half-chair (2H3) conformations for the cyclohexene fragment, while the covalently linked intermediate adopts a flattened half-chair (2H3) conformation. Hybrid QM/MM calculations confirm the structural and electronic properties of the enzyme-bound species and provide insight into key interactions in the enzyme-active site. These insights should stimulate the design of mechanism-based glycoside hydrolase inhibitors with tailored chemical properties.

Highlights

Of note, many glycoside hydrolases (GHs) inhibitors incorporate a basic nitrogen atom[6] that mimics the nascent charge of pyranosylium ion-like transition states[10] (Fig. 2a)
Of the GH mechanisms that catalyze glycosidic bond hydrolysis, most that occur with retention of stereochemistry rely on two active site aspartic acid (Asp) and/or glutamic acid (Glu) residues (Fig. 2a)[1,10,17,18]
We present the structural characterization of our carbasugars with TmGalA in the form of Michaelis complexes for the inhibitor, the reaction products, and most critically, the covalent adduct formed during the turnover of our cyclohexene mimic of 2-deoxy-2fluorogalactose

Summary

Introduction

Many GH inhibitors incorporate a basic nitrogen atom[6] that mimics the nascent charge of pyranosylium ion-like transition states[10] (Fig. 2a). Of the GH mechanisms that catalyze glycosidic bond hydrolysis, most that occur with retention of stereochemistry rely on two active site aspartic acid (Asp) and/or glutamic acid (Glu) residues (Fig. 2a)[1,10,17,18] These enzymes employ sequential SN2like reactions, each involving an inversion of configuration, where the first generates a covalent glycosyl-enzyme intermediate and the second one hydrolyzes the intermediate[1,10,19,20,21]. We present the structural characterization of our carbasugars with TmGalA in the form of Michaelis complexes for the inhibitor (with an active site mutant), the reaction products, and most critically, the covalent adduct formed during the turnover of our cyclohexene mimic of 2-deoxy-2fluorogalactose. Using a combination of quantum mechanics/ molecular mechanics (QM/MM) methods, we provide support for the X-ray structural results by localizing and characterizing the relevant enzyme-bound states

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Results

Conclusion