Amino acid residues forming the active site of arylsulfatase A - Role in catalytic activity and substrate binding

Waldow A, Schmidt B, Dierks T, Bulow von R, Figura von K. Amino acid residues forming the active site of arylsulfatase A - Role in catalytic activity and substrate binding. JOURNAL OF BIOLOGICAL CHEMISTRY. 1999;274(18):12284-12288.Arylsulfatase A belongs to the sulfatase family whose members carry a C alpha-formylglycine that is post-translationally generated by oxidation of a conserved cysteine or serine residue. The formylglycine acts as an aldehyde hydrate with two geminal hydroxyls being involved in catalysis of sulfate ester cleavage. In arylsulfatase A and N-acetylgalactosamine 4-sulfatase this formylglycine was found to form the active site together with a divalent cation and a number of polar residues, tightly interconnected by a net of hydrogen bonds. Most of these putative active site residues are highly conserved among the eukaryotic and prokaryotic members of the sulfatase family. To analyze their function in binding and cleaving sulfate esters, we substituted a total of nine putative active site residues of human ASA by alanine (Asp(29), Asp(30), Asp(281), Asn(282), His(125), His(229), Lys(123), Lys(302), and Ser(150)). In addition the Mg2+-complexing residues (Asp(29), Asp(30), Asp(281), and Asn(282)) were substituted conservatively by either asparagine or aspartate, In all mutants V-max was decreased to 1-26% of wild type activity. The K-m was more than 10-fold increased in K123A and K302A and up to B-fold in the other mutants, In all mutants the pH optimum was increased from 4.5 by 0.2-0.8 units. These results indicate that each of the nine residues examined is critical for catalytic activity, Lys(123) and Lys(302) by binding the substrate and the others by direct (His(125) and Asp(281)) Or indirect participation in catalysis. The shift in the pH optimum is explained by two deprotonation steps that have been proposed for sulfate ester cleavage

1.3 angstrom structure of arylsulfatase from Pseudomonas aeruginosa establishes the catalytic mechanism of sulfate ester cleavage in the sulfatase family

Boltes I, Czapinska H, Kahnert A, et al. 1.3 angstrom structure of arylsulfatase from Pseudomonas aeruginosa establishes the catalytic mechanism of sulfate ester cleavage in the sulfatase family. STRUCTURE. 2001;9(6):483-491.Background: Sulfatases constitute a family of enzymes with a highly conserved active site region including a C alpha -formylglycine that is posttranslationally generated by the oxidation of a conserved cysteine or serine residue. The crystal structures of two human arylsulfatases, ASA and ASB, along with ASA mutants and their complexes led to different proposals for the catalytic mechanism in the hydrolysis of sulfate esters. Results: The crystal structure of a bacterial sulfatase from Pseudomonas aeruginosa (PAS) has been determined at 1.3 Angstrom. Fold and active site region are strikingly similar to those of the known human sulfatases. The structure allows a precise determination of the active site region, unequivocally showing the presence of a C alpha -formylglycine hydrate as the key catalytic residue. Furthermore, the cation located in the active site is unambiguously characterized as calcium by both its B value and the geometry of its coordination sphere. The active site contains a noncovalently bonded sulfate that occupies the same position as the one in para-nitrocate-cholsulfate in previously studied ASA complexes. Conclusions: The structure of PAS shows that the resting state of the key catalytic residue in sulfatases is a formylglycine hydrate. These structural data establish a mechanism for sulfate ester cleavage involving an aldehyde hydrate as the functional group that initiates the reaction through a nucleophilic attack on the sulfur atom in the substrate. The alcohol is eliminated from a reaction intermediate containing pentacoordinated sulfur. Subsequent elimination of the sulfate regenerates the aldehyde, which is again hydrated. The metal cation involved in stabilizing the charge and anchoring the substrate during catalysis is established as calcium

Defective oligomerization of arylsulfatase A as a cause of its instability in lysosomes and metachromatic leukodystrophy

Bulow von R, Schmidt B, Dierks T, et al. Defective oligomerization of arylsulfatase A as a cause of its instability in lysosomes and metachromatic leukodystrophy. JOURNAL OF BIOLOGICAL CHEMISTRY. 2002;277(11):9455-9461.In one of the most common mutations causing metachromatic leukodystrophy, the P426L-allele of arylsulfatase A (ASA), the deficiency of ASA results from its instability in lysosomes. Inhibition of lysosomal cysteine proteinases protects the P426L-ASA and restores the sulfatide catabolism in fibroblasts of the patients. P426L-ASA, but not wild type ASA, was cleaved by purified cathepsin L at threonine 421 yielding 54- and 9-kDa fragments. X-ray crystallography at 2.5-Angstrom resolution showed that cleavage is not due to a difference in the protein fold that would expose the peptide bond following threonine 421 to proteases. Octamerization, which depends on protonation of Glu-424, was impaired for P426L-ASA. The mutation lowers the pH for the octamer/ dimer equilibrium by 0.6 pH units from pH 5.8 to 5.2. A second oligomerization mutant (ASA-A464R) was generated that failed to octamerize even at pH 4.8. A464R-ASA was degraded in lysosomes to catalytically active 54-kDa intermediate. In cathepsin L-deficient fibroblasts, degradation of P426L-ASA and A464R-ASA to the 54-kDa fragment was reduced, while further degradation was blocked. This indicates that defective oligomerization of ASA allows degradation of ASA to a catalytically active 54-kDa intermediate by lysosomal cysteine proteinases, including cathepsin L. Further degradation of the 54-kDa intermediate critically depends on cathepsin L and is modified by the structure of the 9-kDa cleavage product

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

Mariappan M, Radhakrishnan K, Dierks T, Schmidt B, von Figura K. ERp44 mediates a thiol-independent retention of formylglycine-generating enzyme in the endoplasmic reticulum. JOURNAL OF BIOLOGICAL CHEMISTRY. 2008;283(10):6375-6383.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

Posttranslational modification of serine to formylglycine in bacterial sulfatases - Recognition of the modification motif by the iron-sulfur protein AtsB

Marquordt C, Fang QH, Will E, Peng JH, Figura von K, Dierks T. Posttranslational modification of serine to formylglycine in bacterial sulfatases - Recognition of the modification motif by the iron-sulfur protein AtsB. JOURNAL OF BIOLOGICAL CHEMISTRY. 2003;278(4):2212-2218.Calpha-formylglycine is the catalytic residue of sulfatases. Formylglycine is generated by posttranslational modification of a cysteine (pro- and eukaryotes) or serine (pro-karyotes) located in a conserved (C/S)XPXR motif. The modifying enzymes are unknown. AtsB, an iron-sulfur protein, is strictly required for modification of Se-72 in the periplasmic sulfatase AtsA of Klebsiella pneumoniae. Here we show W that AtsB is a cytosolic protein acting on newly synthesized serine-type sulfatases, (ii) that AtsB-mediated FGly formation is dependent on AtsA's signal peptide, and (iii) that the cytosolic cysteine-type sulfatase of Pseudomonas aeruginosa can be converted into a substrate of AtsB if the cysteine is substituted by serine and a signal peptide is added. Thus, formylglycine formation in serine-type sulfatases depends both on AtsB and on the presence of a signal peptide, and AtsB can act on sulfatases of other species. AtsB physically interacts with AtsA in a Ser(72)-dependent manner, as shown in yeast two-hybrid and GST pulldown experiments. This strongly suggests that AtsB is the serine-modifying enzyme and that AtsB relies on a cytosolic function of the sulfatase's signal peptide

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

Dierks T, Schmidt B, Borissenko LV, et al. Multiple sulfatase deficiency is caused by mutations in the gene encoding the human C-alpha-formylglycine generating enzyme. CELL. 2003;113(4):435-444.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

Molecular basis of multiple sulfatase deficiency, mucolipidosis II/III and Niemann-Pick C1 disease - Lysosomal storage disorders caused by defects of non-lysosomal proteins

Dierks T, Schlotawa L, Frese M-A, Radhakrishnan K, von Figura K, Schmidt B. Molecular basis of multiple sulfatase deficiency, mucolipidosis II/III and Niemann-Pick C1 disease - Lysosomal storage disorders caused by defects of non-lysosomal proteins. BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR CELL RESEARCH. 2009;1793(4):710-725.Multiple sulfatase deficiency (MSD), mucolipidosis (MIL) II/III and Niemann-Pick type C1 (NPC1) disease are rare but fatal lysosomal storage disorders caused by the genetic defect of non-lysosomal proteins. The NPC1 protein mainly localizes to late endosomes and is essential for cholesterol redistribution from endocytosed LDL to cellular membranes. NPC1 deficiency leads to lysosomal accumulation of a broad range of lipids. The precise functional mechanism of this membrane protein, however, remains puzzling. ML II, also termed I cell disease. and the less severe ML III result from deficiencies of the Golgi enzyme N-acetylglucosamine 1-phosphotransferase leading to a global defect of lysosome biogenesis. In patient cells, newly synthesized lysosomal proteins are not equipped with the critical lysosomal trafficking marker mannose 6-phosphate, thus escaping from lysosomal sorting at the trans Golgi network. MSD affects the entire sulfatase family, at least seven members of which are lysosomal enzymes that are specifically involved in the degradation of sulfated glycosaminoglycans, sulfolipids or other sulfated molecules. The combined deficiencies of all sulfatases result from a defective post-translational modification by the ER-localized formylglycine-generating enzyme (FGE), which oxidizes a specific cysteine residue to formylglycine, the catalytic residue enabling a unique mechanism of sulfate ester hydrolysis. This review gives an update on the molecular bases of these enigmatic diseases, which have been challenging researchers since many decades and so far led to a number of surprising findings that give deeper insight into both the cell biology and the pathobiochemistry underlying these complex disorders. In case of MSD, considerable progress has been made in recent years towards an understanding of disease-causing FGE mutations. First approaches to link molecular parameters with clinical manifestation have been described and even therapeutical options have been addressed. Further. the discovery of FGE as an essential sulfatase activating enzyme has considerable impact on enzyme replacement or gene therapy of lysosomal storage disorders caused by single sulfatase deficiencies. (C) 2008 Elsevier B.V. All rights reserved