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.posttranslational modification ͉ oxygenase ͉ enzyme mechanism S ulfatases catalyze the hydrolysis of sulfate esters such as glycosaminoglycans, sulfolipids, and steroid sulfates in eukaryotic cells. The key catalytic residue in sulfatases is a unique formylglycine (FGly), which is generated from a cysteine precursor (Fig. 1a) and functions as a nucleophilic aldehyde hydrate in the initial addition reaction of sulfate ester hydrolysis (1). Inactivity of individual sulfatases in humans may lead to severe diseases such as mucopolysaccharidoses, metachromatic leukodystrophy, X-linked ichthyosis, and chondrodysplasia punctata. However, a severe reduction or complete lack of all sulfatase activities, termed multiple sulfatase deficiency, originates from mutations in the FGlygenerating enzyme (FGE) (2, 3).FGE is localized in the endoplasmic reticulum (ER) and modifies the unfolded form of newly synthesized sulfatases (4, 5). The generation of FGly from a cysteine residue is a multistep redox process that involves disulfide bond formation and requires a reducing agent (6) and molecular oxygen (J. Peng, B. Schmidt, A. Preusser-Kunze, M. Mariappan, K. von Figura, and T. Dierks, personal communication) but does not require any cofactors or metal ions. Peptides that contain the minimal motif C-[TSAC]-PSR with flanking sequences according to human sulfatases are FGE substrates and are converted to the...

Molecular analysis ofSUMF1 mutations: stability and residual activity of mutant formylglycine-generating enzyme determine disease severity in multiple sulfatase deficiency

“…This does not preclude that additional factors could influence FGE function and, in case of MSD, act as disease modifiers. Further detailed structural and functional investigations of FGE are required to understand the complex mechanisms of ER retention, turnover regulation, high-affinity binding of a large ensemble of newly synthesized sulfatase polypeptides, for which an unfolded state has to be maintained and, most important, FGly generation through a novel oxygenase mechanism involving a reducing cofactor Roeser et al, 2006]. This knowledge will certainly promote straightforward approaches for designing therapeutic strategies for this fatal disorder.…”

X-ray structure of an AdoMet radical activase reveals an anaerobic solution for formylglycine posttranslational modification

“…Further, in the aerobic system, only four of the 12 hydrogen bonds between FGE and its substrate use the substrate backbone [Protein Data Bank (PDB) ID code 2AIJ]. The majority of interactions are made between peptide sidechains and the maturase, explaining the high sequence specificity in this system (7).…”

“…The maturation of these sulfatases involves two classes of enzymes, one that requires molecular oxygen and another that can function in its absence. FGly generating enzymes (FGEs), found in eukaryotes or aerobically living prokaryotes, generate FGly by oxidizing a cysteine residue on the target sulfatase using molecular oxygen (6,7), whereas anaerobic sulfatase maturating enzymes (anSMEs) generate FGly from either cysteine or serine residues on their target sulfatases using S-adenosyl-L-methionine (AdoMet) radical chemistry (8)(9)(10)(11). In addition to their importance for sulfatase chemistry, FGEs have commercial applications for generating site-specific "aldehyde tags" to use in proteinlabeling technology (12).…”

Partial cure of established disease in an animal model of metachromatic leukodystrophy after intracerebral adeno-associated virus-mediated gene transfer

“…32,33 Coexpression of FGE and ARSA enzyme might enhance the therapeutic effect of ARSA gene delivery. 34 As we used an AAV vector encoding the human ARSA cDNA to treat the ARSA knockout mice, subtle differences in posttranslational modifications between the human and the mouse ARSA enzyme and non-optimal activation of ARSA by FGE enzyme could also have contributed to the lack of complete correction in ARSA knockout mice treated at a symptomatic stage.…”