Cysteine Nucleophiles in Glycosidase Catalysis: Application of a Covalent β-l-Arabinofuranosidase Inhibitor

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 beta-l-arabinofuranosidase inhibitor, hypothesised to react with the catalytic nucleophile to form a non-hydrolysable adduct analogous to the mechanistic covalent intermediate. This beta-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.

Automated summary

The discovery of zinc-dependent retaining glycoside hydrolases presents unresolved mechanistic questions, particularly the controversy surrounding the proposed mechanism involving a zinc-coordinated cysteine nucleophile and a thioglycosyl enzyme intermediate. A synthesized beta-l-arabinofuranosidase inhibitor reacts exclusively with the cysteine thiol catalytic nucleophiles of GH families 127 and 146, supporting further research into the breakdown of the thioglycosyl enzyme intermediate. X-ray crystal structures and simulations provide insight into the mechanisms of zinc-coordinated cysteine as the catalytic nucleophile and the energetics of this unique metalloenzyme clan.