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Cysteine nucleophiles in glycosidase catalysis: Application of a covalent β‐l‐arabinofuranosidase inhibitor

DOI: 10.1002/anie.202013920 DOI Help

Authors: Nicholas G. S. Mcgregor (The University of York) , Joan Coines (Universitat de Barcelona) , Valentina Borlandelli (Leiden University) , Satoko Amaki (The University of Tokyo) , Marta Artola (Leiden University) , Alba Nin‐hill (Universitat de Barcelona) , Daniël Linzel (Leiden University) , Chihaya Yamada (The University of Tokyo) , Takatoshi Arakawa (The University of Tokyo) , Akihiro Ishiwata (RIKEN Cluster for Pioneering Research) , Yukishige Ito (RIKEN Cluster for Pioneering Research; Osaka University) , Gijsbert A. Marel (Leiden University) , Jeroen D. C. Codée (Leiden University) , Shinya Fushinobu (The University of Tokyo) , Herman S. Overkleeft (Leiden University) , Carme Rovira (Universitat de Barcelona; Institució Catalana de Recerca i Estudis Avançats (ICREA)) , Gideon J. Davies (The University of York)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Angewandte Chemie International Edition , VOL 6

State: Published (Approved)
Published: February 2021
Diamond Proposal Number(s): 18598

Abstract: The recent discovery of zinc‐dependent retaining glycoside hydrolases (GHs), with active sites built around a Zn(Cys)3(Glu) coordination complex, has presented unresolved mechanistic questions. In particular, the proposed mechanism, depending on a Zn‐coordinated cysteine nucleophile and passing through a thioglycosyl enzyme intermediate, remains controversial. This is primarily due to the expected stability of the intermediate C−S bond. To facilitate the study of this atypical mechanism, we report the synthesis of a cyclophellitol‐derived β‐l‐arabinofuranosidase inhibitor, hypothesised to react with the catalytic nucleophile to form a non‐hydrolysable adduct analogous to the mechanistic covalent intermediate. This β‐l‐arabinofuranosidase inhibitor reacts exclusively with the proposed cysteine thiol catalytic nucleophiles of representatives of GH families 127 and 146. X‐ray crystal structures determined for the resulting adducts enable MD and QM/MM simulations, which provide insight into the mechanism of thioglycosyl enzyme intermediate breakdown. Leveraging the unique chemistry of cyclophellitol derivatives, the structures and simulations presented here support the assignment of a zinc‐coordinated cysteine as the catalytic nucleophile and illuminate the finely tuned energetics of this remarkable metalloenzyme clan.

Subject Areas: Chemistry, Biology and Bio-materials

Instruments: I03-Macromolecular Crystallography

Other Facilities: NW12A at KEK‐PF

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