Purification, cloning, and characterization of an arylsulfotransferase from the anaerobic bacterium Eubacterium rectale IIIH

Goldberg, S L; Nanduri, Venkata; Cino, P M; Patel, Ramesh N.

doi:10.1038/sj.jim.7000084

Cited by 5 publications

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“…Ni 2ϩ inhibited the enzyme but the inactivated enzyme activity was not reactivated by dialysis. These results suggest that the low specificity activity of the recombinant ASST may be due to the difference of ASST activity assay between the present and previous reports 1,4) or the inactivation of the overexpressed ASST by unknown factors.…”

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confidence: 53%

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Cloning, Expression and Purification of Arylsulfate Sulfotransferase from Eubacterium A-44

Kim

Hyun

Lee

et al. 2007

Biological & Pharmaceutical Bulletin

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Arylsulfate sulfotransferase (ASST) was purified from Eubacterium A-44, 1,2) which is an anaerobe from the human intestinal microflora that metabolizes picosulfate sodium. 3)ASST catalyzes the transfer of a sulfate group from various phenyl sulfate esters to phenolic acceptor substrates. Although Goldberg et al. cloned the ASST gene (astA) from Eubacterium rectale IIIH, the physiological action of ASST is not completely understood.4) Nevertheless, the enzyme has potential use in the in vitro O-sulfation of peptide hormones that require the sulfoconjugation of a hydroxyl group from tyrosine for their biological activity, such as cholecystokinin and hirudin. 5)In this study, the astA gene encoding ASST of Eubacterium A-44 was cloned and compared with the previously reported recombinant ASST in order to determine its use industrially. The cloned astA was expressed in Escherichia coli, and its substrate specificity from the purified recombinant clones was determined. MATERIALS AND METHODS Construction of Eubacterium A-44 Genomic LibraryThe following sequence was used to clone the astA gene of Eubacterium A-44. First, ASST was purified according to previously reported methods, 1) and two internal amino acid peptides, AST 16-1 (5Ј-ACRGCRGARACRGGRTARCA-3Ј) and AST 16-2 (5Ј-CGRTARTARTTNCCNGGNACRTG-3Ј) that were obtained by trypsin digestion were sequenced. The two primers were synthesized according to their sequences and PCR was performed. The PCR product was labeled with DIG and used as a hybridization probe. In order to prepare the genomic library of Eubacterium A-44, the chromosomal DNA was partially digested with HindIII at 37°C for 2 h and size-fractionated on a 0.5% TAE agarose gel. A portion of the digested chromosomal fragments (2-3 kb) was removed from the gel. The DNA was extracted and ligated to the pKF3 vector (Takara, U.S.A.), which was digested with the same enzyme, and transformed into E. coli TH2 competent cells (Takara, U.S.A.). The positive clones were screened.Southern blotting and DNA sequencing were used to confirm the cloned ASST gene.Expression and purification of Eubacterium A-44 ASST In order to express the astA gene, four primers for the full length of ASST, which contain a signal peptide or/and stop codon, were synthesized and amplified by PCR. Two 5Ј primers (BM-A44-F1 and BM-A44-F2) contained a common unique BamHI site: BM-A44-F1 (5Ј-GAGAGAGGATCC-NATGTCAGTAAAATA-3Ј) has a signal peptide but BM-A44-F2(5Ј-GAGAGAGGATCCNGAAGCAGAGCA-3Ј) does not. Two 3Ј primers (BM-A44-R1 and BM-A44-R2) have a common unique HindIII site: BM-A44-R1 (5Ј-GAGAGAAAGCTTGCAGCTGATCGTA-3Ј) does not have a stop codon whereas BM-A44-R2 (5Ј-GAGAGAAAG-CTTTTAGCAGCTGATCGTA-3Ј) does. Each PCR product, which contained a BamHI site at the 5Ј ends and a HindIII site at the 3Ј ends, was subcloned into the pGEM-T easy vector (Promega, U.S.A.), and transformed into the E. coli JM109 competent cells (Promega, U.S.A.). The recombinant plasmid was isolated using a plasmid SV kit (Generalbio, Korea), purified from 0.8% agarose gel and lig...

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“…The specific activity of these purified ASSTs was low compared with that of the previously reported Eubacterium A-44. 4) In order to determine the significance of this observation, the effect of Ni 2ϩ on the ASST activity was investigated. Ni 2ϩ inhibited the enzyme but the inactivated enzyme activity was not reactivated by dialysis.…”

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confidence: 99%

Cloning, Expression and Purification of Arylsulfate Sulfotransferase from Eubacterium A-44

Kim

Hyun

Lee

et al. 2007

Biological & Pharmaceutical Bulletin

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“…Their physiological function remains unclear. With the exception of one enzyme exhibiting relatively low (26%) identity to AstA (Goldberg et al , 2000), all characterized prokaryotic arylsulphotransferases are periplasmic enzymes, and it has been speculated that they play a role in the detoxification of xenobiotic phenolic compounds in the mammalian gut (Baek et al , 1996). No evidence has yet been given on the regulation of their expression as part of the bacterial sulphur cycle.…”

Section: Discussionmentioning

confidence: 99%

The LysR‐type regulator SftR is involved in soil survival and sulphate ester metabolism in Pseudomonas putida

Kahnert

Mirleau

Wait

et al. 2002

Environmental Microbiology

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Sulphate esters make up a large proportion of the available sulphur in agricultural soils, and many pseudomonads can desulphurize a range of aryl- and alkylsulphate esters to provide sulphur for growth. After miniTn5 transposon mutagenesis of Pseudomonas putida S-313, we isolated 19 mutants that were defective in cleavage of the chromogenic sulphate ester 5-bromo-4-chloro-3-indoxylsulphate (X-sulphate). Analysis of these strains revealed that they carried independent insertions in a gene cluster that comprised genes for a sulphate ester/sulphonate transporter (atsRBC) a LysR-type regulator (sftR), an oxygenolytic alkylsulphatase (atsK), an arylsulphotransferase (astA) and a putative TonB-dependent receptor (sftP). The SftP protein was localized in the outer membrane, and the arylsulfphotransferase was identified as an intracellular enzyme. Expression of sftR was repressed in the presence of inorganic sulphate, and the sftR gene was required for the expression of atsBC, atsRK and sftP-astA. An sftR mutant was unable to grow with aryl- or alkylsulphate esters in laboratory media and showed significantly reduced survival compared with the parent strain during incubation in Danish agricultural and grassland soils. This effect suggests that sulphate esters are an important sulphur source for microbes in aerobic soils and highlights the importance of the microbial population in the soil sulphur cycle.

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“…Enzymes possessing the ability to transfer sulfate groups on dierent nucleophiles have been found in bacteria isolated from the intestinal tract of mammals and fecal sources (Baek et al, 1998;Goldberg et al, 2000;Kobashi et al, 1986). The corresponding genes coding for sulfotransferases from the bacteria Klebsiella (Baek et al, 1996) and Eubacterium rectale (Goldberg et al, 2000) has been sequenced and cloned in convenient bacterial hosts with overexpression. As distinct from many mammalian and plant sulfotransferases, catalytic activity of these bacterial enzymes does not require utilization of PAPS.…”

Section: Discussionmentioning

confidence: 99%

“…Puri®cation followed a conventional approach as described in Materials and Methods. A similar procedure has been applied for puri®cation of AST from other bacterial species, Klebsiella and Eubacterium rectale (Goldberg et al, 2000). Results of puri®cation are summarized in Table II.…”

Section: Puri®cation Of Ast From Clostridium Innocuum 554wtmentioning

confidence: 99%

Arylsulfotransferase from Clostridium innocuum—A new enzyme catalyst for sulfation of phenol‐containing compounds

Mozhaev

Khmelnitsky

Sanchez‐Riera

et al. 2002

Biotech & Bioengineering

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Arylsulfotransferase (AST, EC 2.8.2.22), an enzyme capable of sulfating a wide range of phenol-containing compounds was purified from a Clostridium innocuum isolate (strain 554). The enzyme has a molecular weight of 320 kDa and is composed of four subunits. Unlike many mammalian and plant arylsulfotransferases, AST from Clostridium utilizes arylsulfates, including p-nitrophenyl sulfate, as sulfate donors, and is not reactive with 3-phosphoadenosine-5'-phosphosulfate (PAPS). The enzyme possesses broad substrate specificity and is active with a variety of phenols, quinones and flavonoids, but does not utilize primary and secondary alcohols and sugars as substrates. Arylsulfotransferase tolerates the presence of 10 vol% of polar cosolvents (dimethyl formamide, acetonitrile, methanol), but loses significant activity at higher solvent concentrations of 30-40 vol%. The enzyme retains high arylsulfotransferase activity in biphasic systems composed of water and nonpolar solvents, such as cyclohexane, toluene and chloroform, while in biphasic systems with more polar solvents (ethyl acetate, 2-pentanone, methyl tert-butyl ether, and butyl acetate) the enzyme activity is completely lost. High yields of AST-catalyzed sulfation were achieved in reactions with several phenols and tyrosine-containing peptides. Overall, AST studied in this work is a promising biocatalyst in organic synthesis to afford efficient sulfation of phenolic compounds under mild reaction conditions.

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Purification, cloning, and characterization of an arylsulfotransferase from the anaerobic bacterium Eubacterium rectale IIIH

Cited by 5 publications

References 0 publications

Cloning, Expression and Purification of Arylsulfate Sulfotransferase from Eubacterium A-44

Cloning, Expression and Purification of Arylsulfate Sulfotransferase from Eubacterium A-44

The LysR‐type regulator SftR is involved in soil survival and sulphate ester metabolism in Pseudomonas putida

Arylsulfotransferase from Clostridium innocuum—A new enzyme catalyst for sulfation of phenol‐containing compounds

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