Structural basis for covalent inhibition of sulfatases by sulfamate warheads

Abstract

The enzymatic removal of sulfate groups regulates processes ranging from steroid metabolism to carbohydrate degradation. Most sulfatases belong to the S1 family, whose members use a co-translationally installed formylglycine residue to hydrolyse sulfate esters. Arylsulfamates are potent covalent inhibitors of aryl and steroid sulfatases, including the clinical steroid sulfatase inhibitor Irosustat, yet the structure and stability of the inhibited complex remain unresolved. Arylsulfamates and carbohydrate sulfamates do not covalently inhibit many S1 carbohydrate sulfatases despite conservation of their sulfate-binding sites and formylglycine residue. Using enzyme kinetics, X-ray crystallography, molecular dynamics simulations and density functional theory calculations, we define the basis of these contrasting behaviours. High-resolution structures of the Pseudomonas aeruginosa arylsulfatase PaAtsA treated with two arylsulfamates reveal a long-lived tetrahedral, O-linked -hydroxysulfamate adduct attached to formylglycine. Molecular simulations show that replacing sulfate with sulfamate disrupts the favourable Ca2+-oxyanion interaction and alters ligand binding geometry. The permissive hydrophobic binding site of PaAtsA accommodates this rearrangement while retaining a trajectory compatible with nucleophilic attack. By contrast, in the Bacteroides thetaiotaomicron carbohydrate sulfatase BT16363S-Gal, sulfate-to-sulfamate substitution weakens binding and displaces the sulfamate from a reactive pose near the catalytic nucleophile due to a restrictive active site with conserved sugar binding. These findings define the structure and persistence of the arylsulfamate-derived covalent intermediate and explain why sulfamate warheads are tolerated by aryl sulfatases but not carbohydrate sulfatases.