Publications

• A structural and biochemical basis for PAPS-independent sulfuryl transfer by aryl sulfotransferase from uropathogenic Escherichia coli.

  Malojcic G., Owen R.L., Grimshaw J.P., Brozzo M.S., Dreher-Teo H., Glockshuber R.

  Sulfotransferases are a versatile class of enzymes involved in numerous physiological processes. In mammals, adenosine 3'-phosphate-5'-phosphosulfate (PAPS) is the universal sulfuryl donor, and PAPS-dependent sulfurylation of small molecules, including hormones, sugars, and antibiotics, is a criti ... >> More

  Sulfotransferases are a versatile class of enzymes involved in numerous physiological processes. In mammals, adenosine 3'-phosphate-5'-phosphosulfate (PAPS) is the universal sulfuryl donor, and PAPS-dependent sulfurylation of small molecules, including hormones, sugars, and antibiotics, is a critical step in hepatic detoxification and extracellular signaling. In contrast, little is known about sulfotransferases in bacteria, which make use of sulfurylated molecules as mediators of cell-cell interactions and host-pathogen interactions. Bacterial arylsulfate sulfotransferases (also termed aryl sulfotransferases), in contrast to PAPS-dependent sulfotransferases, transfer sulfuryl groups exclusively among phenolic compounds in a PAPS-independent manner. Here, we report the crystal structure of the virulence factor arylsulfate sulfotransferase (ASST) from the prototypic, pyelonephritogenic Escherichia coli strain CFT073 at 2.0-A resolution, and 2 catalytic intermediates, at 2.1-A and 2.4-A resolution, with substrates bound in the active site. ASST is one of the largest periplasmic enzymes and its 3D structure differs fundamentally from all other structurally characterized sulfotransferases. Each 63.8-kDa subunit of the ASST homodimer comprises a 6-bladed beta-propeller domain and a C-terminal beta-sandwich domain. The active sites of the dimer are situated at the center of the channel formed by each beta-propeller and are defined by the side chains of His-252, His-356, Arg-374, and His-436. We show that ASST follows a ping-pong bi-bi reaction mechanism, in which the catalytic residue His-436 undergoes transient sulfurylation, a previously unreported covalent protein modification. The data provide a framework for understanding PAPS-independent sulfotransfer and a basis for drug design targeting this bacterial virulence factor. << Less

  Proc. Natl. Acad. Sci. U.S.A. 105:19217-19222(2008) [PubMed] [EuropePMC]

• Purification, characterization and reaction mechanism of novel arylsulfotransferase obtained from an anaerobic bacterium of human intestine.

  Kim D.H., Konishi L., Kobashi K.

  A novel type of arylsulfotransferase was purified from Eubacterium A-44, one of the predominant bacteria of human intestine. The enzyme (Mr 315 000) was composed of four identical subunits (Mr 80 000) whose N-terminal amino acids were arginine. pI and optimal pH of the enzyme were 3.9 and 8-9, res ... >> More

  A novel type of arylsulfotransferase was purified from Eubacterium A-44, one of the predominant bacteria of human intestine. The enzyme (Mr 315 000) was composed of four identical subunits (Mr 80 000) whose N-terminal amino acids were arginine. pI and optimal pH of the enzyme were 3.9 and 8-9, respectively. The apparent Km for p-nitrophenylsulfate using tyramine as an acceptor substrate and that for tyramine using p-nitrophenylsulfate as a donor substrate were determined to be 0.104 mM and 3.5 mM, respectively. The reaction mechanism of the enzyme was proposed as follows: a donor substrate, p-nitrophenyl [35S]sulfate, combines a histidine residue of the enzyme active site with concomitant release of a phenolic compound, p-nitrophenol. The sulfate group of the histidine residue transfers to a tyrosine group, and then to an acceptor with the binding of another donor to the histidine residue. << Less

  Biochim Biophys Acta 872:33-41(1986) [PubMed] [EuropePMC]

• Cloning, expression and purification of arylsulfate sulfotransferase from Eubacterium A-44.

  Kim B., Hyun Y.J., Lee K.S., Kobashi K., Kim D.H.

  A gene (astA) encoding arylsulfate sulfotransferase (ASST), which transfers a sulfate group from phenolic sulfate esters to phenolic acceptors, was cloned from a Eubacterium A-44 genomic library. The probe (1.5 kb fragment) for the astA gene was prepared from the PCR product of the primers produce ... >> More

  A gene (astA) encoding arylsulfate sulfotransferase (ASST), which transfers a sulfate group from phenolic sulfate esters to phenolic acceptors, was cloned from a Eubacterium A-44 genomic library. The probe (1.5 kb fragment) for the astA gene was prepared from the PCR product of the primers produced using two internal amino acid sequences of ASST, which had been purified from Eubacterium A-44. The astA gene was cloned into the pKF3 vector. Its sequence revealed a 1863 bp open reading frame (ORF) encoding a protein containing 620 amino acids with a secretary signal peptide, and showed 91% homology (identity) to Eubacterium rectale IIIH previously reported. The cloned astA gene was expressed under the T7 promoter of the expression vectors, pET-39b(+) and pET-26b(+), in Escherichia coli BL21 (DE3), and the expressed ASSTs were purified using His Bind column chromatography. The specific activities of the purified ASSTs were 25.6 micromol/min/mg and 37.1 micromol/min/mg, respectively. << Less

  Biol Pharm Bull 30:11-14(2007) [PubMed] [EuropePMC]

• Enzymatic sulfation of quercetin by arylsulfotransferase from a human intestinal bacterium.

  Koizumi M., Shimizu M., Kobashi K.

  A novel type of arylsulfotransferase was partially purified from human intestinal bacteria and its enzymatic properties were examined. Polyphenols such as chalcone, xanthone and flavonoid were found to be sulfated by the bacterial arylsulfotransferase though the sulfation activity varied depending ... >> More

  A novel type of arylsulfotransferase was partially purified from human intestinal bacteria and its enzymatic properties were examined. Polyphenols such as chalcone, xanthone and flavonoid were found to be sulfated by the bacterial arylsulfotransferase though the sulfation activity varied depending upon the positions of the hydroxyl groups. Quercetin, as an example of a flavonol, was rapidly sulfated when p-nitrophenyl sulfate (PNS) was taken as a donor substrate. At a ten-fold molar excess of PNS over quercetin, two products, the 3,3'-disulfate and 3,3',7-trisulfate derivatives, were formed, but the 4'- and 5-hydroxyl groups were not sulfated. In the case of equimolar or two-fold molar excess of PNS to quercetin, only the 3,3'-disulfate was produced and no monosulfate was formed. The enzymatic procedure is useful as a specific and convenient method for the preparation of polyphenol sulfate esters. << Less

  Chem Pharm Bull (Tokyo) 38:794-796(1990) [PubMed] [EuropePMC]

• Purification and reaction mechanism of arylsulfate sulfotransferase from Haemophilus K-12, a mouse intestinal bacterium.

  Lee N.S., Kim B.T., Kim D.H., Kobashi K.

  A novel type of sulfotransferase, arylsulfate sulfotransferase [EC 2.8.2.22], was purified to homogeneity from Haemophilus K-12, a mouse intestinal bacterium. The purified enzyme (M(r) 290,000) is composed of four subunits (M(r) 70,000). The best donor substrate was 4-methylumbelliferyl sulfate, f ... >> More

  A novel type of sulfotransferase, arylsulfate sulfotransferase [EC 2.8.2.22], was purified to homogeneity from Haemophilus K-12, a mouse intestinal bacterium. The purified enzyme (M(r) 290,000) is composed of four subunits (M(r) 70,000). The best donor substrate was 4-methylumbelliferyl sulfate, followed by beta-naphthyl sulfate, p-nitrophenyl sulfate (PNS), and alpha-naphthyl sulfate. The best acceptor substrate was alpha-naphthol, followed by phenol and resorcinol. The apparent Km for PNS using phenol as an acceptor and that for phenol and resorcinol. The apparent Km for PNS using phenol as an acceptor and that for phenol using PNS as a donor substrate were determined to be 0.095 and 0.71 mM, respectively. One of the reaction products, p-nitrophenol inhibited the enzyme noncompetitively with respect to PNS, but competitively with respect to alpha-naphthol. The Ki values of PNP for PNS and alpha-naphthol were 0.89 and 0.12 mM, respectively. The other reaction product, alpha-naphthyl sulfate, inhibited the enzyme competitively with respect to PNS, but non-competitively with respect to alpha-naphthol. The Ki values of alpha-naphthyl sulfate for PNS and for alpha-naphthol were 2.72 and 1.7 mM. These results suggest that the sulfate transfer reaction proceeds according to a ping pong bi bi mechanism. << Less

  J Biochem 118:796-801(1995) [PubMed] [EuropePMC]

• Sulfation of phenolic antibiotics by sulfotransferase obtained from a human intestinal bacterium.

  Kim D.H., Yoon H.K., Koizumi M., Kobashi K.

  Novel sulfotransferase which was isolated from Eubacterium A-44, a human intestinal bacterium, sulfated phenolic antibiotics, such as amoxicillin, ceaodroxil and cefoperazone. The Km values of sulfotransferase for these antibiotics were 6.9, 4.3 and 22.2 mM, respectively. The Vmax values were 8.3, ... >> More

  Novel sulfotransferase which was isolated from Eubacterium A-44, a human intestinal bacterium, sulfated phenolic antibiotics, such as amoxicillin, ceaodroxil and cefoperazone. The Km values of sulfotransferase for these antibiotics were 6.9, 4.3 and 22.2 mM, respectively. The Vmax values were 8.3, 3.3 and 1.6 mumol/min/mg protein. The optimal pH of the enzyme was 9.0, a weakly alkaline region. The antibacterial activity of amoxicillin was not altered by enzymic sulfation of the phenolic hydroxyl group. << Less

  Chem Pharm Bull (Tokyo) 40:1056-1057(1992) [PubMed] [EuropePMC]

• Enzymatic sulfation of polyphenols related to tannins by arylsulfotransferase.

  Koizumi M., Akao T., Kadota S., Kikuchi T., Okuda T., Kobashi K.

  This report discusses a novel type of arylsulfotransferase (AST) which was derived from human intestinal bacterium sulfated polyphenolic compounds when p-nitrophenyl sulfate (PNS) was taken as a donor substrate. (+)-Catechin, (+/-)-catechin, (-)-epicatechin and (-)-epicatechin gallate were better ... >> More

  This report discusses a novel type of arylsulfotransferase (AST) which was derived from human intestinal bacterium sulfated polyphenolic compounds when p-nitrophenyl sulfate (PNS) was taken as a donor substrate. (+)-Catechin, (+/-)-catechin, (-)-epicatechin and (-)-epicatechin gallate were better substrates than tyramine. (-)-Epigallocatechin and (-)-epigallocatechin gallate were slightly worse substrates than tyramine. Although gallic acid was a bad substrate, alkyl gallate esters were better substrates than tyramine. The degree of acceptor specificity increased in proportion to the length of the alkyl group up to the carbon number of five. Pedunculagin, geraniin and corilagin were less effective than tyramine. Rosmarinic acid and penta-O-galloyl-beta-D-glucose were similarly well sulfated. Two products, 4'-monosulfate and 4',5-disulfate of (+)-catechin, were detected at a two-fold molar excess of PNS over (+)-catechin. When (+)-catechin-4'-monosulfate as an acceptor was enzymatically sulfated with PNS as a donor, only the 4',5-disulfate was produced. Thus, arylsulfotransferase was useful for the convenient preparation of sulfate esters of polyphenols at their specific hydroxyl groups. << Less

  Chem Pharm Bull (Tokyo) 39:2638-2643(1991) [PubMed] [EuropePMC]

• Enzymatic sulfation of glycosides and their corresponding aglycones by arylsulfate sulfotransferase from a human intestinal bacterium.

  Konishi-Imamura L., Sato M., Dohi K., Kadota S., Namba T., Kobashi K.

  A novel type of arylsulfate sulfotransferase (ASST) from a predominant human intestinal bacterium catalyzes the stoichiometric transfer of a sulfate group from phenolic sulfate esters to phenols. We clarified that polyphenols were better substrates of this enzyme than the corresponding glycosides. ... >> More

  A novel type of arylsulfate sulfotransferase (ASST) from a predominant human intestinal bacterium catalyzes the stoichiometric transfer of a sulfate group from phenolic sulfate esters to phenols. We clarified that polyphenols were better substrates of this enzyme than the corresponding glycosides. Additionally, a coumarin derivative, esculetin, was sulfated by ASST at the 6-position to give 6-monosulfate. Therefore, ASST is more useful for the preparation of sulfated polyphenols at their specific hydroxyl groups and would play an important role in the metabolism of phenolic compounds in vegetable food and traditional medicines. << Less

  Biol Pharm Bull 17:1018-1022(1994) [PubMed] [EuropePMC]