Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.6.1 (sulfatase)
3,205 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Formation of depurinating adducts by reaction of catechol estrogen-3,4-quinones with DNA was proposed to be a tumor initiating event by estrogens [E.L. Cavalieri et al. (1997) Proc. Natl Acad. Sci. USA, 94, 10937-10942]. Under estrogenic imbalance, oxidation of catechol estrogens to quinones may compete with their detoxification by protective enzymes. The quinones formed can be detoxified by reaction with glutathione (GSH) or can covalently bind to DNA. To provide more support for this hypothesis, we developed a method to identify and quantify GSH, cysteine (Cys) and N-acetylCys conjugates of 4-hydroxyestrogens (4-OHE) in the kidneys of male Syrian hamsters treated with 4-hydroxyestradiol (4-OHE2) by intraperitoneal injection. The highest level of conjugates was observed 1 h after treatment, and almost none was detected after 24 h. Dose-response studies indicated conjugate formation after treatment with 0.5 micromol of 4-OHE2/100 g body weight, and formation increased up to a treatment level of 12 micromol/100 g body weight. GSH, Cys and N-acetylCys conjugates of 4-OHE were identified in the picomole range by high-performance liquid chromatography (HPLC) with multichannel electrochemical detection and confirmed by HPLC/tandem mass spectrometry. Treatment of tissue homogenates with beta-glucuronidase/sulfatase at 37 degrees C for 6 h before extraction resulted in a 12- to 20-fold increase in Cys conjugates from picomole to nanomole levels. Similar enhancement was observed by just incubating the tissue at 37 degrees C for 6 h. Evidence for the 4-OHE-1-N7Gua depurinating adducts was obtained by mass spectrometry. We conclude that GSH and Cys conjugates of the 4-OHE and the 4-OHE-N7Gua adducts can be utilized as biomarkers to detect estrogenic imbalance and potential susceptibility to tumor initiation.
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PMID:Catechol estrogen conjugates and DNA adducts in the kidney of male Syrian golden hamsters treated with 4-hydroxyestradiol: potential biomarkers for estrogen-initiated cancer. 1123 91

Sulfatases are members of a highly conserved family of enzymes that catalyze the hydrolysis of sulfate ester bonds from a variety of substrates. The functional correlation reflects a high degree of amino acid sequence similarity along the entire length, in particular in the active site where the C(X)PSR consensus sequence is present. Cysteine undergoes an important co- or post-translation modification essential for the accomplishment of catalytic activity: conversion in formylglycine. In this work, the cysteine of heparan N-sulfatase (NS) was replaced either by a serine (C70S) or by a methionine (C70M) using site-directed mutagenesis. C70S and C70M mutant cDNAs were expressed and analyzed in COS cells; both mutations caused a loss of NS activity; however, while C70S showed a normal precursor form undergoing processing to a reduced mature form within the lysosomes, C70M was poorly synthesized and formed a complex with the molecular chaperone immunoglobulin binding protein.
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PMID:Heparan N-sulfatase: cysteine 70 plays a role in the enzyme catalysis and processing. 1157 45

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.
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PMID:Characterization of posttranslational formylglycine formation by luminal components of the endoplasmic reticulum. 1160 May 3

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-A 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.
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PMID:Defective oligomerization of arylsulfatase a as a cause of its instability in lysosomes and metachromatic leukodystrophy. 1177 24

Calpha-formylglycine is the catalytic residue of sulfatases. Formylglycine is generated by posttranslational modification of a cysteine (pro- and eukaryotes) or serine (prokaryotes) located in a conserved (C/S)XPXR motif. The modifying enzymes are unknown. AtsB, an iron-sulfur protein, is strictly required for modification of Ser(72) in the periplasmic sulfatase AtsA of Klebsiella pneumoniae. Here we show (i) 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 pull-down 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.
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PMID:Posttranslational modification of serine to formylglycine in bacterial sulfatases. Recognition of the modification motif by the iron-sulfur protein AtsB. 1241 7

In the previous paper (Myette, J. R., Shriver, Z., Claycamp, C., McLean, M. W., Venkataraman, G., and Sasisekharan, R. (2003) J. Biol. Chem. 278, 12157-12166), we described the molecular cloning, recombinant expression, and preliminary biochemical characterization of the heparin/heparan sulfate 2-O-sulfatase from Flavobacterium heparinum. In this paper, we extend our structure-function investigation of the 2-O-sulfatase. First, we have constructed a homology-based structural model of the enzyme active site, using as a framework the available crystallographic data for three highly related arylsulfatases. In this model, we have identified important structural parameters within the enzyme active site relevant to enzyme function, especially as they relate to its substrate specificity. By docking various disaccharide substrates, we identified potential structural determinants present within these substrates that would complement this unique active site architecture. These determinants included the position and number of sulfates present on the glucosamine, oligosaccharide chain length, the presence of a Delta4,5-unsaturated double bond, and the exolytic versus endolytic potential of the enzyme. The predictions made from our model provided a structural basis of substrate specificity originally interpreted from the biochemical and kinetic data. Our modeling approach was further complemented experimentally using peptide mapping in tandem with mass spectrometry and site-directed mutagenesis to physically demonstrate the presence of a covalently modified cysteine (formylglycine) within the active site. This combinatorial approach of structure modeling and biochemical studies provides insight into the molecular basis of enzyme function.
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PMID:The heparin/heparan sulfate 2-O-sulfatase from Flavobacterium heparinum. A structural and biochemical study of the enzyme active site and saccharide substrate specificity. 1251 74

Heparan sulfate glycosaminoglycans are structurally complex polysaccharides critically engaged in a wide range of cell and tissue functions. Any structure-based approach to study their respective biological functions is facilitated by the use of select heparan sulfate glycosaminoglycan-degrading enzymes with unique substrate specificities. We recently reported of one such enzyme, the Delta4,5-glycuronidase cloned from Flavobacterium heparinum and recombinantly expressed in Escherichia coli (Myette, J. R., Shriver, Z., Kiziltepe, T., McLean, M. W., Venkataraman, G., and Sasisekharan, R. (2002) Biochemistry 41, 7424-7434). In this study, we likewise report the molecular cloning of the 2-O-sulfatase from the same bacterium and its recombinant expression as a soluble, highly active enzyme. At the protein level, the flavobacterial 2-O-sulfatase possesses considerable sequence homology to other members of a large sulfatase family, especially within its amino terminus, where the highly conserved sulfatase domain is located. Within this domain, we have identified by sequence homology the critical active site cysteine predicted to be chemically modified as a formylglycine in vivo. We also present a characterization of the biochemical properties of the enzyme as it relates to optimal in vitro reaction conditions and a kinetic description of its substrate specificity. In particular, we demonstrate that in addition to the fact that the enzyme exclusively hydrolyzes the sulfate at the 2-O-position of the uronic acid, it also exhibits a kinetic preference for highly sulfated glucosamines within each disaccharide unit, especially those possessing a 6-O-sulfate. The sulfatase also displays a clear kinetic preference for disaccharides with beta1-->4 linkages but is able, nevertheless, to hydrolyze unsaturated, 2-O-sulfated chondroitin disaccharides. Finally, we describe the substrate-product relationship of the 2-O-sulfatase to the Delta4,5-glycuronidase and the analytical value of using both of these enzymes in tandem for elucidating heparin/heparan sulfate composition.
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PMID:The heparin/heparan sulfate 2-O-sulfatase from Flavobacterium heparinum. Molecular cloning, recombinant expression, and biochemical characterization. 1251 75

C(alpha)-Formylglycine, the catalytic amino acid residue in the active site of sulfatases, is generated by post-translational modification of a cysteine or serine residue. We describe a highly sensitive procedure for the detection of C(alpha)-formylglycine-containing peptides in tryptic digests of sulfatase proteins. The protocol is based on the formation of hydrazone derivatives of C(alpha)-formylglycine-containing peptides when using dinitrophenylhydrazine as a matrix for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). The hydrazone derivatives desorb and ionize with high efficiency and can be detected in the sub-femtomole range. The presence of C(alpha)-formylglycine is indicated by a mass increment of 180.13 u, corresponding to the hydrazone moiety, and also by a unique C-terminal fragment ion, characteristic of sulfatases, that becomes prominent in MALDI post-source decay mass spectra of the hydrazone derivatives.
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PMID:Identification of formylglycine in sulfatases by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. 1252 9

We describe three novel mutations in the human arylsulfatase A gene in three patients with MLD, an autosomal recessive lysosomal storage disorder. An insertion, 2590_2591insCCCC in exon 8 and a deletion, 752_758delGCCGGCC, in exon 3 will both result in frameshifts. A mutation in exon 8, 2566T-->C, results in a missense mutation C488R, disrupting an unusual cysteine-knot at the C-terminal end of the protein. All three mutations are heterozygous with previously documented mutations. A previously reported mutation, R84Q was identified on a pseudodeficiency allele. These mutations are part of a heterogeneous spectrum of mutations found in a collection of DNA samples from MLD patients from across Canada and the USA.
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PMID:Spectrum of mutations in the arylsulfatase A gene in a Canadian DNA collection including two novel frameshift mutations, a new missense mutation (C488R) and an MLD mutation (R84Q) in cis with a pseudodeficiency allele. 1280 38

Recently, the human C(alpha)-formylglycine (FGly)-generating enzyme (FGE), whose deficiency causes the autosomal-recessively transmitted lysosomal storage disease multiple sulfatase deficiency (MSD), has been identified. In sulfatases, FGE posttranslationally converts a cysteine residue to FGly, which is part of the catalytic site and is essential for sulfatase activity. FGE is encoded by the sulfatase modifying factor 1 (SUMF1) gene, which defines a new gene family comprising orthologs from prokaryotes to higher eukaryotes. The genomes of E. coli, S. cerevisiae and C. elegans lack SUMF1, indicating a phylogenetic gap and the existence of an alternative FGly-generating system. The genomes of vertebrates including mouse, man and pufferfish contain a sulfatase modifying factor 2 (SUMF2) gene encoding an FGE paralog of unknown function. SUMF2 evolved from a single exon SUMF1 gene as found in diptera prior to divergent intron acquisition. In several prokaryotic genomes, the SUMF1 gene is cotranscribed with genes encoding sulfatases which require FGly modification. The FGE protein contains a single domain that is made up of three highly conserved subdomains spaced by nonconserved sequences of variable lengths. The similarity among the eukaryotic FGE orthologs varies between 72% and 100% for the three subdomains and is highest for the C-terminal subdomain, which is a hotspot for mutations in MSD patients.
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PMID:The human SUMF1 gene, required for posttranslational sulfatase modification, defines a new gene family which is conserved from pro- to eukaryotes. 1456 51


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