A general binding mechanism for all human sulfatases by the formylglycine-generating enzyme

Abstract

The formylglycine (FGly)-generating enzyme (FGE) uses molecular oxygen to oxidize a conserved cysteine residue in all eukaryotic sulfatases to the catalytically active FGly. Sulfatases degrade and remodel sulfate esters, and inactivity of FGE results in multiple sulfatase deficiency, a fatal disease. The previously determined FGE crystal structure revealed two crucial cysteine residues in the active site, one of which was thought to be implicated in substrate binding. The other cysteine residue partakes in a novel oxygenase mechanism that does not rely on any cofactors. Here, we present crystal structures of the individual FGE cysteine mutants and employ chemical probing of wild-type FGE, which defined the cysteines to differ strongly in their reactivity. This striking difference in reactivity is explained by the distinct roles of these cysteine residues in the catalytic mechanism. Hitherto, an enzyme-substrate complex as an essential cornerstone for the structural evaluation of the FGly formation mechanism has remained elusive. We also present two FGE-substrate complexes with pentamer and heptamer peptides that mimic sulfatases. The peptides isolate a small cavity that is a likely binding site for molecular oxygen and could host reactive oxygen intermediates during cysteine oxidation. Importantly, these FGE-peptide complexes directly unveil the molecular bases of FGE substrate binding and specificity. Because of the conserved nature of FGE sequences in other organisms, this binding mechanism is of general validity. Furthermore, several disease-causing mutations in both FGE and sulfatases are explained by this binding mechanism.

Fig. 1.

Overall fold of FGE and comparison of wild-type FGE (PDB entry 1Y1I) with the active site mutants C336S and C341S, and the IAM-modified wild-type FGE. (a) Scheme of the reaction catalyzed by FGE. (b) Ribbon representation of FGE with the cysteine residues drawn as stick models and structural Ca²⁺ ions displayed as magenta and cyan spheres. Cys-336 and Cys-341 are part of the active site. (c–f) Close-ups of the region around Cys-336 and Cys-341. (c) Cys336Ser mutant. (d) Cys341Ser mutant with Cys-336 oxidized to the sulfonic acid (Ocs). (e) Superposition of reduced wild-type FGE (yellow) with the Cys336Ser mutant (blue) showing the minor effect of the mutation on the structure. (f) Incubation of wild-type FGE with IAM leads exclusively to carboxamidomethylation of Cys-336 (Acm), whereas Cys-341 remains unaffected. The region Tyr-340–Cys-341 shows two alternate conformations, which are shown in yellow and blue. All σ_A-weighted mFo-DFc omit electron density maps, including Fig. 2c, are contoured at 3σ.

Fig. 2.

Substrate binding to FGE. (a) The surface representation of FGE shows a groove with the redox-active cysteine pair Cys-336/Cys-341 (red surface) at one end. Pro-182 (green surface) marks the site of a cross-link with a photoreactive substrate peptide (7) and hence is also close to the substrate binding site (8). (b) FGE–peptide complex. The peptide LCTPSRA binds to Cys-341 via an intermolecular disulfide bond. The FGE surface is colored according to electrostatic potential (±10 kT), showing a negative patch close to the C terminus of the peptide, which is neutralized by Arg-P73. (c) Close-up of b rotated 45° clockwise showing the exquisite surface complementarity of the peptide with FGE.

Fig. 3.

Substrate binding and mechanistic details. (a) Hydrogen bonds are shown as dashed lines, and water molecules are drawn as red spheres. (b) General binding mechanism of FGE to all human sulfatases. The schematic drawing generalizes the binding of unfolded sulfatases to FGE as the first step in FGly formation. (c) Magnification of the region adjacent to the intermolecular disulfide bond. The orientation of the Tyr-340 side chain in the apo– and peptide–FGE structures differs by 6.4 Å (compare with Fig. 4). Only residues Cys-P69 and Thr-P70 of the peptide are drawn. The solvent-inaccessible volume between the disulfide bond and serine residues 333 and 336 (transparent gray sphere) is occupied by Cl^– (green) in the complex structure. (d) Possible mechanisms after the activation of molecular oxygen by FGE. Atoms from O₂ are indicated in red. A novel hydroperoxide intermediate is formulated from which two alternative avenues for FGly formation are conceivable. Currently, no distinction between these two pathways is possible.