Post-translational formylglycine modification of bacterial sulfatases by the radical S-adenosylmethionine protein AtsB

C(alpha)-Formylglycine (FGly) is the catalytic residue of sulfatases. FGly is generated by post-translational modification of a cysteine (prokaryotes and eukaryotes) or serine (prokaryotes) located in a conserved (C/S)XPXR motif. AtsB of Klebsiella pneumoniae is directly involved in FGly generation from serine. AtsB is predicted to belong to the newly discovered radical S-adenosylmethionine (SAM) superfamily. By in vivo and in vitro studies we show that SAM is the critical co-factor for formation of a functional AtsB.SAM.sulfatase complex and for FGly formation by AtsB. The SAM-binding site of AtsB involves (83)GGE(85) and possibly also a juxtaposed FeS center coordinated by Cys(39) and Cys(42), as indicated by alanine scanning mutagenesis. Mutation of these and other conserved cysteines as well as treatment with metal chelators fully impaired FGly formation, indicating that all three predicted FeS centers are crucial for AtsB function. It is concluded that AtsB oxidizes serine to FGly by a radical mechanism that is initiated through reductive cleavage of SAM, thereby generating the highly oxidizing deoxyadenosyl radical, which abstracts a hydrogen from the serine-C(beta)H(2)-OH side chain.     

The iron sulfur protein AtsB is required for posttranslational formation of formylglycine in the Klebsiella sulfatase.

The catalytic residue of eukaryotic and prokaryotic sulfatases is a alpha-formylglycine. In the sulfatase of Klebsiella pneumoniae the formylglycine is generated by posttranslational oxidation of serine 72. We cloned the atsBA operon of K. pneumoniae and found that the sulfatase was expressed in inactive form in Escherichia coli transformed with the structural gene (atsA). Coexpression of the atsB gene, however, led to production of high sulfatase activity, indicating that the atsB gene product plays a posttranslational role that is essential for the sulfatase to gain its catalytic activity. This was verified after purification of the sulfatase from the periplasm of the cells. Peptide analysis of the protein expressed in the presence of AtsB revealed that half of the polypeptides carried the formylglycine at position 72, while the remaining polypeptides carried the encoded serine. The inactive sulfatase expressed in the absence of AtsB carried exclusively serine 72, demonstrating that the atsB gene is required for formylglycine modification. This gene encodes a 395-amino acid residue iron sulfur protein that has a cytosolic localization and is supposed to directly or indirectly catalyze the oxidation of the serine to formylglycine.     

In vitro characterization of AtsB, a radical SAM formylglycine-generating enzyme that contains three [4Fe-4S] clusters

Sulfatases catalyze the cleavage of a variety of cellular sulfate esters via a novel mechanism that requires the action of a protein-derived formylglycine cofactor. Formation of the cofactor is catalyzed by an accessory protein and involves the two-electron oxidation of a specific cysteinyl or seryl residue on the relevant sulfatase. Although some sulfatases undergo maturation via mechanisms in which oxygen serves as an electron acceptor, AtsB, the maturase from Klebsiella pneumoniae, catalyzes the oxidation of Ser72 on AtsA, its cognate sulfatase, via an oxygen-independent mechanism. Moreover, it does not make use of pyridine or flavin nucleotide cofactors as direct electron acceptors. In fact, AtsB has been shown to be a member of the radical S-adenosylmethionine superfamily of proteins, suggesting that it catalyzes this oxidation via an intermediate 5'-deoxyadenosyl 5'-radical that is generated by a reductive cleavage of S-adenosyl- l-methionine. In contrast to AtsA, very little in vitro characterization of AtsB has been conducted. Herein we show that coexpression of the K. pneumoniae atsB gene with a plasmid that encodes genes that are known to be involved in iron-sulfur cluster biosynthesis yields soluble protein that can be characterized in vitro. The as-isolated protein contained 8.7 +/- 0.4 irons and 12.2 +/- 2.6 sulfides per polypeptide, which existed almost entirely in the [4Fe-4S] (2+) configuration, as determined by Mossbauer spectroscopy, suggesting that it contained at least two of these clusters per polypeptide. Reconstitution of the as-isolated protein with additional iron and sulfide indicated the presence of 12.3 +/- 0.2 irons and 9.9 +/- 0.4 sulfides per polypeptide. Subsequent characterization of the reconstituted protein by Mossbauer spectroscopy indicated the presence of only [4Fe-4S] clusters, suggesting that reconstituted AtsB contains three per polypeptide. Consistent with this stoichiometry, an as-isolated AtsB triple variant containing Cys --> Ala substitutions at each of the cysteines in its CX 3CX 2C radical SAM motif contained 7.3 +/- 0.1 irons and 7.2 +/- 0.2 sulfides per polypeptide while the reconstituted triple variant contained 7.7 +/- 0.1 irons and 8.4 +/- 0.4 sulfides per polypeptide, indicating that it was unable to incorporate an additional cluster. UV-visible and Mossbauer spectra of both samples indicated the presence of only [4Fe-4S] clusters. AtsB was capable of catalyzing multiple turnovers and exhibited a V max/[E T] of approximately 0.36 min (-1) for an 18-amino acid peptide substrate using dithionite to supply the requisite electron and a value of approximately 0.039 min (-1) for the same substrate using the physiologically relevant flavodoxin reducing system. Simultaneous quantification of formylglycine and 5'-deoxyadenosine as a function of time indicates an approximate 1:1 stoichiometry. Use of a peptide substrate in which the target serine is changed to cysteine also gives rise to turnover, supporting approximately 4-fold the activity of that observed with the natural substrate.     

A novel protein modification generating an aldehyde group in sulfatases: its role in catalysis and disease

In multiple sulfatase deficiency, a rare human lysosomal storage disorder, all known sulfatases are synthesized as catalytically poorly active polypeptides. Analysis of the latter has shown that they lack a protein modification that was detected in all members of the sulfatase family. This novel protein modification generates a 2-amino-3-oxopropanoic acid (Cα-formylglycine) residue by oxidation of the thiol group of a cysteine that is conserved among all eukaryotic sulfatases. The oxidation occurs in the endoplasmic reticulum at a stage when the nascent polypeptide is not yet folded. The aldehyde is part of the catalytic site and is likely to act as an aldehyde hydrate. One of the geminal hydroxyl groups accepts the sulfate during sulfate ester cleavage leading to the formation of a covalently sulfated enzyme intermediate. The other hydroxyl is required for the subsequent elimination of the sulfate and regeneration of the aldehyde group. In some prokaryotic members of the sulfatase gene family, the DNA sequence predicts a serine residue, and not a cysteine. Analysis of one of these prokaryotic sulfatases, however, revealed the presence of the Cα-formylglycine indicating that the aldehyde group is essential for all members of the sulfatase family and that it can be generated from either cysteine or serine. BioEssays 20 :505–510, 1998. © 1998 John Wiley & Sons, Inc.     

Sequence determinants directing conversion of cysteine to formylglycine in eukaryotic sulfatases

Sulfatases carry at their catalytic site a unique post-translational modification, an alpha-formylglycine residue that is essential for enzyme activity. Formylglycine is generated by oxidation of a conserved cysteine or, in some prokaryotic sulfatases, serine residue. In eukaryotes, this oxidation occurs in the endoplasmic reticulum during or shortly after import of the nascent sulfatase polypeptide. The modification of arylsulfatase A was studied in vitro and was found to be directed by a short linear sequence, CTPSR, starting with the cysteine to be modified. Mutational analyses showed that the cysteine, proline and arginine are the key residues within this motif, whereas formylglycine formation tolerated the individual, but not the simultaneous substitution of the threonine or serine. The CTPSR motif was transferred to a heterologous protein leading to low-efficient formylglycine formation. The efficiency reached control values when seven additional residues (AALLTGR) directly following the CTPSR motif in arylsulfatase A were present. Mutating up to four residues simultaneously within this heptamer sequence inhibited the modification only moderately. AALLTGR may, therefore, have an auxiliary function in presenting the core motif to the modifying enzyme. Within the two motifs, the key residues are fully, and other residues are highly conserved among all known members of the sulfatase family.     

A new type of bacterial sulfatase reveals a novel maturation pathway in prokaryotes

Sulfatases are a highly conserved family of enzymes found in all three domains of life. To be active, sulfatases undergo a unique post-translational modification leading to the conversion of either a critical cysteine ("Cys-type" sulfatases) or a serine ("Ser-type" sulfatases) into a Calpha-formylglycine (FGly). This conversion depends on a strictly conserved sequence called "sulfatase signature" (C/S)XPXR. In a search for new enzymes from the human microbiota, we identified the first sulfatase from Firmicutes. Matrix-assisted laser desorption ionization time-of-flight analysis revealed that this enzyme undergoes conversion of its critical cysteine residue into FGly, even though it has a modified (C/S)XAXR sulfatase signature. Examination of the bacterial and archaeal genomes sequenced to date has identified many genes bearing this new motif, suggesting that the definition of the sulfatase signature should be expanded. Furthermore, we have also identified a new Cys-type sulfatase-maturating enzyme that catalyzes the conversion of cysteine into FGly, in anaerobic conditions, whereas the only enzyme reported so far to be able to catalyze this reaction is oxygen-dependent. The new enzyme belongs to the radical S-adenosyl-l-methionine enzyme superfamily and is related to the Ser-type sulfatase-maturating enzymes. This finding leads to the definition of a new enzyme family of sulfatase-maturating enzymes that we have named anSME (anaerobic sulfatase-maturating enzyme). This family includes enzymes able to maturate Cys-type as well as Ser-type sulfatases in anaerobic conditions. In conclusion, our results lead to a new scheme for the biochemistry of sulfatases maturation and suggest that the number of genes and bacterial species encoding sulfatase enzymes is currently underestimated.     

First evidences for a third sulfatase maturation system in prokaryotes from E. coli aslB and ydeM deletion mutants

To be active all known arylsulfatases undergo a unique post-translational modification leading to the conversion of an active site residue (serine or cysteine) into a C(alpha)-formylglycine. Although deprived of sulfatase activity, Escherichia coli K12 can efficiently mature heterologous Cys-type sulfatases. Three potential enzymes (AslB, YdeM and YidF) belonging to the anaerobic sulfatase maturating enzyme family (an SME) are present in its genome. Here we show that E. coli could mature Cys-type sulfatases only in aerobic conditions and that knocking-out of aslB, ydeM and yidF does not impair Cys-type sulfatase maturation. These findings demonstrate that these putative anSME are not involved in Cys-type sulfatase maturation and strongly support the existence of a second, oxygen-dependent and Cys-type specific sulfatase maturation system among prokaryotes.     

Characterization of posttranslational formylglycine formation by luminal components of the endoplasmic reticulum

C(alpha)-formylglycine is the key catalytic residue in the active site of sulfatases. In eukaryotes formylglycine is generated during or immediately after sulfatase translocation into the endoplasmic reticulum by oxidation of a specific cysteine residue. We established an in vitro assay that allowed us to measure formylglycine modification independent of protein translocation. The modifying enzyme was recovered in a microsomal detergent extract. As a substrate we used ribosome-associated nascent chain complexes comprising in vitro synthesized sulfatase fragments that were released from the ribosomes by puromycin. Formylglycine modification was highly efficient and did not require a signal sequence in the substrate polypeptide. Ribosome association helped to maintain the modification competence of nascent chains but only after their release efficient modification occurred. The modifying machinery consists of soluble components of the endoplasmic reticulum lumen, as shown by differential extraction of microsomes. The in vitro assay can be performed under kinetically controlled conditions. The activation energy for formylglycine formation is 61 kJ/mol, and the pH optimum is approximately 10. The activity is sensitive to the SH/SS equilibrium and is stimulated by Ca(2+). Formylglycine formation is efficiently inhibited by a synthetic sulfatase peptide representing the sequence directing formylglycine modification. The established assay system should make possible the biochemical identification of the modifying enzyme.     

Function and structure of a prokaryotic formylglycine-generating enzyme

Type I sulfatases require an unusual co- or post-translational modification for their activity in hydrolyzing sulfate esters. In eukaryotic sulfatases, an active site cysteine residue is oxidized to the aldehyde-containing C(alpha)-formylglycine residue by the formylglycine-generating enzyme (FGE). The machinery responsible for sulfatase activation is poorly understood in prokaryotes. Here we describe the identification of a prokaryotic FGE from Mycobacterium tuberculosis. In addition, we solved the crystal structure of the Streptomyces coelicolor FGE homolog to 2.1 A resolution. The prokaryotic homolog exhibits remarkable structural similarity to human FGE, including the position of catalytic cysteine residues. Both biochemical and structural data indicate the presence of an oxidized cysteine modification in the active site that may be relevant to catalysis. In addition, we generated a mutant M. tuberculosis strain lacking FGE. Although global sulfatase activity was reduced in the mutant, a significant amount of residual sulfatase activity suggests the presence of FGE-independent sulfatases in this organism.     

Aryl sulfamates are broad spectrum inactivators of sulfatases: Effects on sulfatases from various sources

Aryl sulfamates were originally developed as inhibitors of steroid sulfatase, and have recently been shown to be powerful inactivators of a bacterial sulfatase, PaAtsA from Pseudomonas aeruginosa. We demonstrate that a simple aryl sulfamate, 3-nitrophenyl sulfamate, can inactivate sulfatases from various sources including snail, limpet and abalone. In each case inactivation was time-dependent and active-site directed, as demonstrated by protection against inactivation by substrate. These results suggest that such easily acquired aryl sulfamates can be used as reliable biochemical reagents for the study of sulfatases from a diverse array of sources.     

SUMF1 enhances sulfatase activities in vivo in five sulfatase deficiencies

Sulfatases are enzymes that hydrolyse a diverse range of sulfate esters. Deficiency of lysosomal sulfatases leads to human diseases characterized by the accumulation of either GAGs (glycosaminoglycans) or sulfolipids. The catalytic activity of sulfatases resides in a unique formylglycine residue in their active site generated by the post-translational modification of a highly conserved cysteine residue. This modification is performed by SUMF1 (sulfatase-modifying factor 1), which is an essential factor for sulfatase activities. Mutations in the SUMF1 gene cause MSD (multiple sulfatase deficiency), an autosomal recessive disease in which the activities of all sulfatases are profoundly reduced. In previous studies, we have shown that SUMF1 has an enhancing effect on sulfatase activity when co-expressed with sulfatase genes in COS-7 cells. In the present study, we demonstrate that SUMF1 displays an enhancing effect on sulfatases activity when co-delivered with a sulfatase cDNA via AAV (adeno-associated virus) and LV (lentivirus) vectors in cells from individuals affected by five different diseases owing to sulfatase deficiencies or from murine models of the same diseases [i.e. MLD (metachromatic leukodystrophy), CDPX (X-linked dominant chondrodysplasia punctata) and MPS (mucopolysaccharidosis) II, IIIA and VI]. The SUMF1-enhancing effect on sulfatase activity resulted in an improved clearance of the intracellular GAG or sulfolipid accumulation. Moreover, we demonstrate that the SUMF1-enhancing effect is also present in vivo after AAV-mediated delivery of the sulfamidase gene to the muscle of MPSIIIA mice, resulting in a more efficient rescue of the phenotype. These results indicate that co-delivery of SUMF1 may enhance the efficacy of gene therapy in several sulfatase deficiencies.     

Cloning of a mucin-desulfating sulfatase gene from Prevotella strain RS2 and its expression using a Bacteroides recombinant system

A gene encoding the mucin-desulfating sulfatase in Prevotella strain RS2 has been cloned, sequenced, and expressed in an active form. A 600-bp PCR product generated using primers designed from amino acid sequence data was used to isolate a 5,058-bp genomic DNA fragment containing the mucin-desulfating sulfatase gene. A 1,551-bp open reading frame encoding the sulfatase proprotein was identified, and the deduced 517-amino-acid protein minus its signal sequence corresponded well with the published mass of 58 kDa estimated by denaturing gel electrophoresis. The sulfatase sequence showed homology to aryl- and nonarylsulfatases with different substrate specificities from the sulfatases of other organisms. No sulfatase activity could be detected when the sulfatase gene was cloned into Escherichia coli expression vectors. However, cloning the gene into a Bacteroides expression vector did produce active sulfatase. This is the first mucin-desulfating sulfatase to be sequenced and expressed. A second open reading frame (1,257 bp) was identified immediately upstream from the sulfatase gene, coding in the opposite direction. Its sequence has close homology to iron-sulfur proteins that posttranslationally modify other sulfatases. By analogy, this protein is predicted to catalyze the modification of a serine group to a formylglycine group at the active center of the mucin-desulfating sulfatase, which is necessary for enzymatic activity.     

Expression, localization, structural, and functional characterization of pFGE, the paralog of the Calpha-formylglycine-generating enzyme

pFGE is the paralog of the formylglycine-generating enzyme (FGE), which catalyzes the oxidation of a specific cysteine to Calpha-formylglycine, the catalytic residue in the active site of sulfatases. The enzymatic activity of sulfatases depends on this posttranslational modification, and the genetic defect of FGE causes multiple sulfatase deficiency. The structural and functional properties of pFGE were analyzed. The comparison with FGE demonstrates that both share a tissue-specific expression pattern and the localization in the lumen of the endoplasmic reticulum. Both are retained in the endoplasmic reticulum by a saturable mechanism. Limited proteolytic cleavage at similar sites indicates that both also share a similar three-dimensional structure. pFGE, however, is lacking the formylglycine-generating activity of FGE. Although overexpression of FGE stimulates the generation of catalytically active sulfatases, overexpression of pFGE has an inhibitory effect. In vitro pFGE interacts with sulfatase-derived peptides but not with FGE. The inhibitory effect of pFGE on the generation of active sulfatases may therefore be caused by a competition of pFGE and FGE for newly synthesized sulfatase polypeptides.     

ERp44 mediates a thiol-independent retention of formylglycine-generating enzyme in the endoplasmic reticulum

Inside the endoplasmic reticulum (ER) formylglycine-generating enzyme (FGE) catalyzes in newly synthesized sulfatases the post-translational oxidation of a specific cysteine. Thereby formylglycine is generated, which is essential for sulfatase activity. Here we show that ERp44 interacts with FGE forming heterodimeric and, to a lesser extent, also heterotetrameric and octameric complexes, which are stabilized through disulfide bonding between cysteine 29 of ERp44 and cysteines 50 and 52 in the N-terminal region of FGE. ERp44 mediates FGE retrieval to the ER via its C-terminal RDEL signal. Increasing ERp44 levels by overexpression enhances and decreasing ERp44 levels by silencing reduces ER retention of FGE. Suppressing disulfide bonding by mutating the critical cysteines neither abrogates ERp44.FGE complex formation nor interferes with ERp44-mediated retention of FGE, indicating that noncovalent interactions between ERp44 and FGE are sufficient to mediate ER retention. The N-terminal region of FGE harboring Cys(50) and Cys(52) is dispensible for catalytic activity in vitro but required for FGE-mediated activation of sulfatases in vivo. This in vivo activity is affected neither by overexpression nor by silencing of ERp44, indicating that a further ER component interacting with the N-terminal extension of FGE is critical for sulfatase activation.     

A major step on the road to understanding a unique posttranslational modification and its role in a genetic disease

The posttranslational conversion of cysteine to C(alpha)-formylglycine in the catalytic site of mammalian sulfatases is deficient in the rare but devastating disorder multiple sulfatase deficiency (MSD). Two papers in this issue of Cell report the cloning of a gene responsible for this activity.     

Monounsaturated fatty acid modification of Wnt protein: its role in Wnt secretion

The secretion and extracellular transport of Wnt protein are thought to be well-regulated processes. Wnt is known to be acylated with palmitic acid at a conserved cysteine residue (Cys77 in murine Wnt-3a), and this residue appears to be required for the control of extracellular transport. Here, we show that murine Wnt-3a is also acylated at a conserved serine residue (Ser209). Of note, we demonstrated that this residue is modified with a monounsaturated fatty acid, palmitoleic acid. Wnt-3a defective in acylation at Ser209 is not secreted from cells in culture or in Xenopus embryos, but it is retained in the endoplasmic reticulum (ER). Furthermore, Porcupine, a protein with structural similarities to membrane-bound O-acyltransferases, is required for Ser209-dependent acylation, as well as for Wnt-3a transport from the ER for secretion. These results strongly suggest that Wnt protein requires a particular lipid modification for proper intracellular transport during the secretory process.     

Cloning and characterization of two cDNAs encoding sulfatases in the Roman snail, Helix pomatia

The sulfatase from the snail Heli pomatia is widely used for analytical applications. We have investigated the content of sulfatases in H. pomatia, using a biochemical and a molecular approach. A 112-kDa protein from the intestinal juice of H. pomatia comigrated with sulfatase activity when chromatographed on Sephacryl S300 and concanavalin A-Sepharose. The N-terminal amino acid sequence of the protein was similar to one of three sulfatase motifs defined by sequence alignment of known sulfatases. Degenerate primers designed from the motifs and the N-terminal amino acid sequence obtained were used to generate PCR fragments and to isolate both a full-length and a 3'-truncated cDNA encoding H. pomatia sulfatases, designated SULF1 and SULF2. SULF1 consists of 503 amino acids and shows 53-55% identity to the mammalian arylsulfatase B. The amino acid sequence deduced from the 878-bp SULF2 cDNA fragment is 55% identical with SULF1. Both SULF1 and SULF2 contain the cysteine residue conserved in the active site of many sulfatases, which is known to be posttranslationally modified into formylglycine in eukaryotic sulfatases. However, the SULF1 and SULF2 cDNAs do not code for the protein purified. This indicates the presence of at least three sulfatase genes in H. pomatia.     

Molecular characterization of the human Calpha-formylglycine-generating enzyme

Calpha-formylglycine (FGly) is the catalytic residue in the active site of sulfatases. In eukaryotes, it is generated in the endoplasmic reticulum by post-translational modification of a conserved cysteine residue. The FGly-generating enzyme (FGE), performing this modification, is an endoplasmic reticulum-resident enzyme that upon overexpression is secreted. Recombinant FGE was purified from cells and secretions to homogeneity. Intracellular FGE contains a high mannose type N-glycan, which is processed to the complex type in secreted FGE. Secreted FGE shows partial N-terminal trimming up to residue 73 without loosing catalytic activity. FGE is a calcium-binding protein containing an N-terminal (residues 86-168) and a C-terminal (residues 178-374) protease-resistant domain. The latter is stabilized by three disulfide bridges arranged in a clamp-like manner, which links the third to the eighth, the fourth to the seventh, and the fifth to the sixth cysteine residue. The innermost cysteine pair is partially reduced. The first two cysteine residues are located in the sequence preceding the N-terminal protease-resistant domain. They can form intramolecular or intermolecular disulfide bonds, the latter stabilizing homodimers. The C-terminal domain comprises the substrate binding site, as evidenced by yeast two-hybrid interaction assays and photocross-linking of a substrate peptide to proline 182. Peptides derived from all known human sulfatases served as substrates for purified FGE indicating that FGE is sufficient to modify all sulfatases of the same species.     

Multiple sulfatase deficiency is caused by mutations in the gene encoding the human C(alpha)-formylglycine generating enzyme

C(alpha)-formylglycine (FGly) is the catalytic residue in the active site of eukaryotic sulfatases. It is posttranslationally generated from a cysteine in the endoplasmic reticulum. The genetic defect of FGly formation causes multiple sulfatase deficiency (MSD), a lysosomal storage disorder. We purified the FGly generating enzyme (FGE) and identified its gene and nine mutations in seven MSD patients. In patient fibroblasts, the activity of sulfatases is partially restored by transduction of FGE encoding cDNA, but not by cDNA carrying an MSD mutation. The gene encoding FGE is highly conserved among pro- and eukaryotes and has a paralog of unknown function in vertebrates. FGE is localized in the endoplasmic reticulum and is predicted to have a tripartite domain structure.