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Cysteine Nucleophiles in Glycosidase Catalysis: Application of a Covalent β‐ l‐ Arabinofuranosidase Inhibitor
Author(s) -
McGregor Nicholas G. S.,
Coines Joan,
Borlandelli Valentina,
Amaki Satoko,
Artola Marta,
NinHill Alba,
Linzel Daniël,
Yamada Chihaya,
Arakawa Takatoshi,
Ishiwata Akihiro,
Ito Yukishige,
Marel Gijsbert A.,
Codée Jeroen D. C.,
Fushinobu Shinya,
Overkleeft Herman S.,
Rovira Carme,
Davies Gideon J.
Publication year - 2021
Publication title -
angewandte chemie
Language(s) - English
Resource type - Journals
eISSN - 1521-3757
pISSN - 0044-8249
DOI - 10.1002/ange.202013920
Subject(s) - nucleophile , chemistry , cysteine , covalent bond , stereochemistry , adduct , catalysis , thiol , combinatorial chemistry , enzyme , organic chemistry
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.