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)

Pseudomonas aeruginosa PAO1 grew in defined synthetic medium with any of a broad variety of single sulfur sources, including sulfate, cysteine, thiocyanate, alkanesulfonates, organosulfate esters and methionine, but not with aromatic sulfonates, thiophenols or organothiocyanates or isothiocyanates. During growth with any of these compounds except sulfate, cysteine or thiocyanate, a set of 10 sulfate starvation-induced (SSI) proteins was strongly up-regulated, as observed by two-dimensional protein electrophoresis of total cell extracts. A comparable level of up-regulation was found for the hydrolytic enzyme arylsulfatase, which has previously been used as a marker enzyme for the sulfate starvation response. One of the SSI proteins was identified by N-terminal sequencing as a high-affinity periplasmic sulfate-binding protein, and another was related to thiol-specific antioxidants, but the N-terminal sequences of the other SSI proteins revealed no similarity to N-termini of proteins of known function, and they probably represent uncharacterized enzymes involved in sulfur scavenging when preferred sulfur sources are absent. To study the role that cysteine biosynthetic intermediates play in the synthesis of these proteins in vivo, we isolated mini-Tn5 transposon mutants of P. aeruginosa with insertions in the cysN and cysI genes, which encode subunits of ATP-sulfurylase and sulfite reductase, respectively. These two genes were cloned and sequenced. cysI showed high similarity to the cognate gene in Escherichia coli, whereas cysN encoded a 69.3 kDa protein with two domains corresponding to the E. coli CysN and CysC proteins. Sulfate no longer repressed synthesis of the SSI proteins in cysN mutants, but repression was restored by sulfite; in the cysI mutant, sulfate, sulfite and sulfide all led to repression of SSI protein synthesis. This suggests that there are at least two independent corepressors of the sulfate starvation response in this species.
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PMID:Regulation of the sulfate starvation response in Pseudomonas aeruginosa: role of cysteine biosynthetic intermediates. 961 12

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 (C alpha-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 C alpha-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.
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PMID:A novel protein modification generating an aldehyde group in sulfatases: its role in catalysis and disease. 969 62

Eukaryotic sulfatases carry an alpha-formylglycine residue that is essential for activity and is located within the catalytic site. This formylglycine is generated by posttranslational modification of a conserved cysteine residue. The arylsulfatase gene of Pseudomonas aeruginosa also encodes a cysteine at the critical position. This protein could be expressed in active form in a sulfatase-deficient strain of P. aeruginosa, thereby restoring growth on aromatic sulfates as sole sulfur source, and in Escherichia coli. Analysis of the mature protein expressed in E. coli revealed the presence of formylglycine at the expected position, showing that the cysteine is also converted to formylglycine in a prokaryotic sulfatase. Substituting the relevant cysteine by a serine codon in the P. aeruginosa gene led to expression of inactive sulfatase protein, lacking the formylglycine. The machinery catalyzing the modification of the Pseudomonas sulfatase in E. coli therefore resembles the eukaryotic machinery, accepting cysteine but not serine as a modification substrate. By contrast, in the arylsulfatase of Klebsiella pneumoniae a formylglycine is found generated by modification of a serine residue. The expression of both the Klebsiella and the Pseudomonas sulfatases as active enzymes in E. coli suggests that two modification systems are present, or that a common modification system is modulated by a cofactor.
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PMID:Posttranslational formation of formylglycine in prokaryotic sulfatases by modification of either cysteine or serine. 974 19

Sulfatases contain a unique posttranslational modification in their active site, a formylglycine residue generated from a cysteine or a serine residue. The formylglycine residue is part of a sequence that is highly conserved among sulfatases, suggesting that it might direct the generation of this unique amino acid derivative. In the present study residues 68-86 flanking formylglycine 69 in arylsulfatase A were subjected to an alanine/glycine scanning mutagenesis. The mutants were analyzed for the conversion of cysteine 69 to formylglycine and their kinetic properties. Only cysteine 69 turned out to be essential for formation of the formylglycine residue, while substitution of leucine 68, proline 71, and alanine 74 within the heptapeptide LCTPSRA reduced the formylglycine formation to about 30-50%. Several residues that are part of or directly adjacent to an alpha-helix presenting the formylglycine 69 at the bottom of the active site pocket were found to be critical for catalysis. A surprising outcome of this study was that a number of residues fully or highly conserved between all known eukaryotic and prokaryotic sulfatases turned out to be essential neither for generation of formylglycine nor for catalysis.
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PMID:Residues critical for formylglycine formation and/or catalytic activity of arylsulfatase A. 976 Feb 28

Enzymatic hydrolysis with beta-glucuronidase/sulfatase was used for the enantioselective determination of N-hydroxymexiletine glucuronide in plasma for pharmacokinetic studies. N-Hydroxymexiletine glucuronide was determined as the quantity of mexiletine released by hydrolysis (difference between the enantiomeric concentrations of mexiletine obtained with and without hydrolysis). Plasma samples (100 microliters) were treated at pH 5.0 with 10 mg of the enzyme (Limpet Acetone Powder type I) for 16 hr at 37 degrees C and extracted at pH 10.4 with diisopropyl ether. Chiral mexiletine discrimination was obtained by reaction with o-phthalaldehyde/N-acetyl-L-cysteine, separation of the resulting diastereomers on a C-18 reversed-phase column with a mobile phase of methanol-0.05 N acetate buffer, pH 5.5 (6.5:3.5, v/v), and fluorescence detection (lambda ex 350 nm, lambda em 455 nm). The performance characteristics for the enantioselective analysis of mexiletine preceded by enzymatic hydrolysis were recovery approximately 90%, quantification limit 1 ng/ml, and linearity up to 1000 ng/ml plasma for both enantiomers. The coefficients of variation obtained in the study of intra- and inter-day precision were respectively 5% and 7% for both enantiomers. The assay was shown to be suitable for a pharmacokinetic study performed in a patient with the arrhythmic form of chronic Chagas' heart disease treated with 200 mg t.i.d. of racemic mexiletine hydrochloride. The high sensitivity of the method allows analysis of only 100 microliters plasma.
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PMID:Enantioselective analysis of N-hydroxymexiletine glucuronide in human plasma for pharmacokinetic studies. 995

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.
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PMID:Sequence determinants directing conversion of cysteine to formylglycine in eukaryotic sulfatases. 1020 63

Arylsulfatase A belongs to the sulfatase family whose members carry a Calpha-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 (Asp29, Asp30, Asp281, Asn282, His125, His229, Lys123, Lys302, and Ser150). In addition the Mg2+-complexing residues (Asp29, Asp30, Asp281, and Asn282) were substituted conservatively by either asparagine or aspartate. In all mutants Vmax was decreased to 1-26% of wild type activity. The Km was more than 10-fold increased in K123A and K302A and up to 5-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, Lys123 and Lys302 by binding the substrate and the others by direct (His125 and Asp281) 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.
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PMID:Amino acid residues forming the active site of arylsulfatase A. Role in catalytic activity and substrate binding. 1021 97

A gene cluster upstream of the arylsulfatase gene (atsA) in Pseudomonas aeruginosa was characterized and found to encode a putative ABC-type transporter, AtsRBC. Mutants with insertions in the atsR or atsB gene were unable to grow with hexyl-, octyl-, or nitrocatecholsulfate, although they grew normally with other sulfur sources, such as sulfate, methionine, and aliphatic sulfonates. AtsRBC therefore constitutes a general sulfate ester transport system, and desulfurization of aromatic and medium-chain-length aliphatic sulfate esters occurs in the cytoplasm. Expression of the atsR and atsBCA genes was repressed during growth with sulfate, cysteine, or thiocyanate. No expression of these genes was observed in the cysB mutant PAO-CB, and the ats genes therefore constitute an extension of the cys regulon in this species.
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PMID:The sulfur-regulated arylsulfatase gene cluster of Pseudomonas aeruginosa, a new member of the cys regulon. 1071 18

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.
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PMID:Cloning and characterization of two cDNAs encoding sulfatases in the Roman snail, Helix pomatia. 1077 44

Arylsulfatase A (ASA) 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 crystal structures of two arylsulfatases, ASA and ASB, and kinetic studies on ASA mutants led to different proposals for the catalytic mechanism in the hydrolysis of sulfate esters. The structures of two ASA mutants that lack the functional C(alpha)-formylglycine residue 69, in complex with a synthetic substrate, have been determined in order to unravel the reaction mechanism. The crystal structure of the inactive mutant C69A-ASA in complex with p-nitrocatechol sulfate (pNCS) mimics a reaction intermediate during sulfate ester hydrolysis by the active enzyme, without the covalent bond to the key side-chain FGly69. The structure shows that the side-chains of lysine 123, lysine 302, serine 150, histidine 229, the main-chain of the key residue 69 and the divalent cation in the active center are involved in sulfate binding. It is proposed that histidine 229 protonates the leaving alcoholate after hydrolysis.C69S-ASA is able to bind covalently to the substrate and hydrolyze it, but is unable to release the resulting sulfate. Nevertheless, the resulting sulfation is low. The structure of C69S-ASA shows the serine side-chain in a single conformation, turned away from the position a substrate occupies in the complex. This suggests that the double conformation observed in the structure of wild-type ASA is more likely to correspond to a formylglycine hydrate than to a twofold disordered aldehyde oxo group, and accounts for the relative inertness of the C69S-ASA mutant. In the C69S-ASA-pNCS complex, the substrate occupies the same position as in the C69A-ASA-pNCS complex, which corresponds to the non-covalently bonded substrate. Based on the structural data, a detailed mechanism for sulfate ester cleavage is proposed, involving an aldehyde hydrate as the functional group.
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PMID:Crystal structure of an enzyme-substrate complex provides insight into the interaction between human arylsulfatase A and its substrates during catalysis. 1112 5


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