Cysteine Nucleophiles in Glycosidase Catalysis: Application of a Covalent β‐l‐Arabinofuranosidase Inhibitor. (2nd February 2021)

• Record Type: Journal Article
• Authors: McGregor, Nicholas G. S.; Coines, Joan; Borlandelli, Valentina; Amaki, Satoko; Artola, Marta; Nin‐Hill, Alba; Linzel, Daniël; Yamada, Chihaya; Arakawa, Takatoshi; Ishiwata, Akihiro; Ito, Yukishige; van der Marel, Gijsbert A.; Codée, Jeroen D. C.; Fushinobu, Shinya; Overkleeft, Herman S.; Rovira, Carme; Davies, Gideon J.
• Is Part Of: Angewandte Chemie. Volume 133:Number 11(2021)
• Page Start: 5818
• Page End: 5822
• Publication Date: 2021-02-02
• Subjects: arabinofuranosidase -- cyclophellitol -- glycoside hydrolase -- metalloenzyme -- zinc; Chemistry -- Periodicals; 540
• DOI: 10.1002/ange.202013920
• Languages: English
• ISSNs: 0044-8249

Abstract

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. Abstract : β‐l ‐arabinofuranosidases are thought to make use of a unique zinc‐associated cysteine nucleophile to catalyze glycoside hydrolysis. Using a bespoke synthetic covalent inhibitor, this proposed mechanism is put to the test. Mass spectrometry, X‐ray crystallography, and mechanistic simulations support the formation of a thioglycosyl enzyme intermediate and reveal how the enzyme facilitates CS bond cleavage.