Molecular Basis for Multiple Sulfatase Deficiency and Mechanism for Formylglycine Generation of the Human Formylglycine-Generating Enzyme

Abstract

Sulfatases are enzymes essential for degradation and remodeling of sulfate esters. Formylglycine (FGly), the key catalytic residue in the active site, is unique to sulfatases. In higher eukaryotes, FGly is generated from a cysteine precursor by the FGly-generating enzyme (FGE). Inactivity of FGE results in multiple sulfatase deficiency (MSD), a fatal autosomal recessive syndrome. Based on the crystal structure, we report that FGE is a single-domain monomer with a surprising paucity of secondary structure and adopts a unique fold. The effect of all 18 missense mutations found in MSD patients is explained by the FGE structure, providing a molecular basis of MSD. The catalytic mechanism of FGly generation was elucidated by six high-resolution structures of FGE in different redox environments. The structures allow formulation of a novel oxygenase mechanism whereby FGE utilizes molecular oxygen to generate FGly via a cysteine sulfenic acid intermediate.

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

“…So far, 30 mutations in SUMF1 have been described in MSD patients including nine nonsense and 21 missense mutations [Dierks et al, 2003;Cosma et al, 2003;Cosma et al, 2004;Dierks et al, 2005;Diaz-Font et al, 2005]. Mature FGE is made up of a 341-amino acid polypeptide, which adopts a single domain monomeric structure with a unique fold .…”

SUMF1 mutations affecting stability and activity of formylglycine generating enzyme predict clinical outcome in multiple sulfatase deficiency

“…13,20,29,21 ECL signals were quantified using the AIDA software package (Raytest, Straubenhardt, Germany). Endogenous FGE in MSD fibroblasts was detected using ECL femto substrate and enhancer diluted 1:10 in ECL solution (Pierce, Bonn, Germany).…”

“…25 Until now, more than 30 different SUMF1 mutations were found, most of which are missense mutations. 9,23,26,20,27 In a first approach, we investigated the consequences of four different MSD causing SUMF1 mutations on FGE 13 expression, localization, stability and activity in either an overexpressing cell-culture model or in patient fibroblasts. We found that all four variant FGE proteins could be expressed in HT-1080 cells, correctly localizing to the ER.…”

“…[17][18][19] This modification occurs late co or early posttranslationally in the endoplasmic reticulum (ER) and is mediated by the FGly-generating enzyme (FGE), an unusual mono-oxygenase using molecular oxygen for oxidation of Cb of the substrate cysteine through its catalytic cysteines 336 and 341. 20,21 FGE is encoded by the sulfatase-modifying factor 1 gene (SUMF1). Patients with MSD carry mutations in this gene.…”

Synthesis of Heterobifunctional Protein Fusions Using Copper‐Free Click Chemistry and the Aldehyde Tag

“…FGE oxidizes the genetically-encoded cysteine residue to the aldehyde-bearing residue formylglycine (fGly) during protein expression in either E. coli or mammalian cells (Scheme 1 A). [35] The aldehyde can then be modified by hydrazone or oxime formation (Scheme 1 B). [36] Thus, the aldehyde tag serves as a means for site-specific introduction of azides or cyclooctynes onto recombinant proteins through small-molScheme 1.…”