Nucleotide Binding and Sulfation Catalyzed by Phenol Sulfotransferase

doi:10.1006/bbrc.2000.2722

Biochemical and Biophysical Research Communications

Volume 271, Issue 3, 19 May 2000, Pages 818-822

https://doi.org/10.1006/bbrc.2000.2722 Get rights and content

Abstract

The sulfation of a nucleotide is an indispensable step for the sulfuryl group transfer in a biological system. The product and cosubstrate of sulfotransferase in physiological condition are adenosine 3′,5′-bisphosphate (PAP) and 3′-phospho adenosine 5′-phosphosulfate (PAPS), respectively. We find that ribose and adenine, two major parts of the adenosine nucleotide, bind tightly to phenol sulfotransferase (PST) separately, and various nucleotides also bind tightly to PST. We determine the dissociation constants of a variety of nucleotides and examine their potential as cofactors or cosubstrates of PST. Using 4-nitrophenyl sulfate as the sulfuryl group donor, three nucleotides, adenosine 5′-monophosphate (AMP), adenosine 2′,5′-bisphosphate (2′,5′-PAP), and adenosine 2′:3′-cyclic phosphate 5′-phosphate (2′:3′-cyclic PAP), are shown here for the first time to be sulfated at 5′-phopho position by a PST catalyzed reaction. Spectrophotometry, HPLC, and ³¹P NMR are used to determine the activity of PST and identify the sulfated nucleotides. The V_max of PST and K_m of these nucleotides are determined when they are used as cofactors or cosubstrates for the sulfuryl group transfer. The existence and possible physiological significance of these newly reported binding and sulfation of nucleotides by PST in biology is yet to be discovered.

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A nano switch mechanism for the redox-responsive sulfotransferase
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Citation Excerpt :
Each titration was performed in triplicate together with controls without the addition of the enzyme or DTNB, respectively. The Kd was determined by fluorescence as described previously [26]. An aliquot amount of PAP was added into the solution containing SULT (0.06 or 1 μM), 50 mM potassium phosphate buffer at pH 7.0.
Cellular redox signaling is important in diverse physiological and pathological processes. The activity of rat phenol sulfotransferase (rSULT1A1), which is important for the metabolism of hormone and drug, is subjected to redox regulation. Two cysteines, Cys232 and Cys66, nanometer away from each other and from the enzyme active site were proposed to form disulfide bond to regulate the activity of rSULT1A1. A nano switch, composed of a flexible loop from amino acid residues 59–70, explained how this long distance interaction between two cysteines can be achieved. The enzyme properties were investigated through site-directed muatagnesis, circular dichroism, enzyme kinetics and homologous modeling of the rSULT1A1 structures. We proposed that the formation of disulfide bond between Cys232 and Cys66 induced conformational changes of sulfotransferase, then in turn affected its nucleotide binding and enzyme activity. This discovery was extended to understand the possible redox regulation of other sulfotransferases from different organisms. The redox switch can be created in other redox-insensitive sulfotransferases, such as human phenol sulfotransferase (hSULT1A1) and human alcohol sulfotransferase (hSULT2A1), to produce mutant enzymes with redox regulation capacity. This study strongly suggested that redox regulation of drug and hormone metabolism can be significantly varied even though the sequence and structure of SULT1A1 of human and rat have a high degree of homology.
Identification and characterization of two novel cytosolic sulfotransferases, SULT1 ST7 and SULT1 ST8, from zebrafish
2008, Aquatic Toxicology
Cytosolic sulfotransferases (SULTs) constitute a family of Phase II detoxification enzymes that are involved in the protection against potentially harmful xenobiotics as well as the regulation and homeostasis of endogenous compounds. Compared with humans and rodents, the zebrafish serves as an excellent model for studying the role of SULTs in the detoxification of environmental pollutants including environmental estrogens. By searching the expressed sequence tag database, two zebrafish cDNAs encoding putative SULTs were identified. Sequence analysis indicated that these two putative zebrafish SULTs belong to the SULT1 gene family. The recombinant form of these two novel zebrafish SULTs, designated SULT1 ST7 and SULT1 ST8, were expressed using the pGEX-2TK glutathione S-transferase (GST) gene fusion system and purified from transformed BL21 (DE3) cells. Purified GST-fusion protein form of SULT1 ST7 and SULT1 ST8 exhibited strong sulfating activities toward environmental estrogens, particularly hydroxylated polychlorinated biphenyls (PCBs), among various endogenous and xenobiotic compounds tested as substrates. pH-dependence experiments showed that SULT1 ST7 and SULT1 ST8 displayed pH optima at 6.5 and 8.0, respectively. Kinetic parameters of the two enzymes in catalyzing the sulfation of catechin and chlorogenic acid as well as 3-chloro-4-biphenylol were determined. Developmental expression experiments revealed distinct patterns of expression of SULT1 ST7 and SULT1 ST8 during embryonic development and throughout the larval stage onto maturity.
Identification of novel hydroxysteroid-sulfating cytosolic SULTs, SULT2 ST2 and SULT2 ST3, from zebrafish: Cloning, expression, characterization, and developmental expression
2006, Archives of Biochemistry and Biophysics
Citation Excerpt :
As a control, PCR amplification of the sequence encoding zebrafish β-actin was concomitantly performed using the above-mentioned first-strand cDNAs as templates, in conjunction with gene-specific sense and antisense oligonucleotide primers (Table 1) designed based on reported zebrafish β-actin nucleotide sequence (GenBank Accession No. AF057040). PAP[35S] was synthesized from ATP and carrier-free [35S]sulfate using the bifunctional human ATP sulfurylase/APS kinase and its purity determined as previously described [22,23]. The PAP[35S] synthesized was adjusted to the required concentration and specific activity by the addition of cold PAPS.
By searching the expressed sequence tag database, two zebrafish cDNAs encoding putative cytosolic sulfotransferases (SULTs) were identified. Sequence analysis indicated that these two zebrafish SULTs belong to the cytosolic SULT2 gene family. The recombinant form of these two novel zebrafish SULTs, designated SULT2 ST2 and SULT2 ST3, were expressed using the pGEX-2TK glutathione S-transferase (GST) gene fusion system and purified from transformed BL21 (DE3) Escherichia coli cells. Purified GST-fusion protein form of SULT2 ST2 and SULT2 ST3 exhibited strong sulfating activities toward dehydroepiandrosterone (DHEA) and corticosterone, respectively, among various endogenous compounds tested as substrates. Both enzymes displayed pH optima at ∼6.5. Kinetic constants of the two enzymes, as well as the GST-fusion protein form of the previously identified SULT2 ST1, with DHEA and corticosterone as substrates were determined. Developmental stage-dependent expression experiments revealed distinct patterns of expression of SULT2 ST2 and SULT2 ST3, as well as the previously identified SULT2 ST1, during embryonic development and throughout the larval stage onto maturity.
Identification of a novel zebrafish SULT1 cytosolic sulfotransferase: Cloning, expression, characterization, and developmental expression study
2005, Archives of Biochemistry and Biophysics
Citation Excerpt :
As a control, PCR amplification of zebrafish β-actin was concomitantly performed using the above-mentioned first-strand cDNAs as templates, in conjunction with gene-specific sense and antisense oligonucleotide primers (cf. Table 1) designed based on reported nucleotide sequence of zebrafish β-actin (GenBank Accession No. AF057040). [35S]PAPS was synthesized from ATP and carrier-free [35S]sulfate using the bifunctional human ATP sulfurylase/APS kinase and its purity was determined as previously described [29,30]. The [35S]PAPS synthesized was then adjusted to the required concentration and specific activity by the addition of cold PAPS.
By searching the zebrafish expressed sequence tag database, we had identified two partial cDNA clones encoding the 5′- and 3′-regions of a putative cytosolic sulfotransferase (SULT). Using the reverse transcription-polymerase chain reaction (RT-PCR) technique, a full-length cDNA encoding this zebrafish SULT was amplified, cloned, and sequenced. Analysis of the sequence data revealed that this novel zebrafish SULT displays 49, 46, and 45% amino acid sequence identity to human SULT1A1, mouse SULT1D1, and rat SULT1C1. This zebrafish SULT therefore appears to belong to the SULT1 cytosolic SULT gene family. Recombinant zebrafish SULT (designated SULT1 isoform 4), expressed using the pGEX-2TK prokaryotic expression vector and purified from transformed Escherichia coli cells, migrated as a 35 kDa protein upon sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Among the endogenous compounds tested as substrates, the purified SULT1 isoform 4 displayed significant sulfating activities toward thyroid hormones, estrone, and dehydroepiandrosterone. The enzyme also showed activities toward a number of xenobiotics including some flavonoids, isoflavonoids, and other phenolic compounds, with a pH optimum at 7.0. A thermostability experiment revealed the enzyme to be relatively stable over a temperature range between 28 and 37 °C. Among 10 divalent metal cations tested, Fe²⁺, Hg²⁺, Co²⁺, Zn²⁺, Cu²⁺, and Cd²⁺ exhibited dramatic inhibitory effects on the activity of the enzyme. Developmental expression study using RT-PCR revealed that the zebrafish SULT1 isoform 4 showed a low level of expression in the segmentation period during the embryonic development, which gradually increased to a high level of expression throughout the larval stage onto maturity.
Identification of a novel estrogen-sulfating cytosolic SULT from zebrafish: Molecular cloning, expression, characterization, and ontogeny study
2005, Biochemical and Biophysical Research Communications
Citation Excerpt :
Miscellaneous methods. PAP[35S] was synthesized from ATP and carrier-free [35S]sulfate using the bifunctional human ATP sulfurylase/adenosine 5′-phosphosulfate kinase and its purity was determined as previously described [18]. The PAP[35S] synthesized was adjusted to the required concentration and specific activity of 15 Ci/mmol at 1.4 mM by the addition of cold PAPS.
By searching the expressed sequence tag database, a zebrafish cDNA encoding a putative cytosolic sulfotransferase (SULT) was identified. Sequence analysis indicated that this zebrafish SULT belongs to the SULT1 cytosolic SULT gene family. The recombinant form of this novel zebrafish SULT, expressed using the pGEX-2TK expression system and purified from transformed BL21 (DE3) Escherichia coli cells, displayed sulfating activities specifically for estrone and 17β-estradiol among various endogenous compounds tested as substrates. The enzyme also exhibited sulfating activities toward some xenobiotic phenolic compounds. This new zebrafish SULT showed dual pH optima, at 6.5 and 10–10.5, with estrone or n-propyl gallate as substrate. Kinetic constants of the sulfation of estrone, 17β-estradiol, and n-propyl gallate were determined. Developmental stage-dependent expression experiments revealed a significant level of expression of this novel zebrafish estrogen-sulfating SULT at the beginning of the hatching period during embryogenesis, which continued throughout the larval stage onto maturity.
cDNA cloning, expression, and functional characterization of a zebrafish SULT1 cytosolic sulfotransferase
2003, Archives of Biochemistry and Biophysics
Using the reverse transcriptase-polymerase chain reaction technique, a full-length cDNA encoding a novel zebrafish sulfotransferase was cloned and sequenced. Sequence analysis indicated that this zebrafish sulfotransferase belongs to the SULT1 cytosolic sulfotransferase gene family. The recombinant form of the zebrafish sulfotransferase, purified from Escherichia coli cells, displayed sulfating activities toward a number of endogenous compounds, in particular dopamine and thyroid hormones, in addition to xenobiotics including some flavonoids, isoflavonoids, and other phenolic compounds. The zebrafish sulfotransferase exhibited substrate dependence in pH optimum. In comparison with those determined with dopamine as substrate, the zebrafish sulfotransferase displayed much lower K_m and higher V_max with n-propyl gallate as substrate. A thermostability experiment revealed the enzyme to be relatively stable over a temperature range between 20 and 43 °C. Among 10 divalent metal cations tested, Hg²⁺, Co²⁺, Zn²⁺, Cd²⁺, Cu²⁺, and Pb²⁺ exhibited dramatic inhibitory effects on the activity of the zebrafish sulfotransferase.

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^☆: Abbreviations used: PAPS, 3′-phospho adenosine 5′-phosphosulfate; PAP, 3′-phospho adenosine 5′-phosphate or adenosine 3′, 5′-bisphosphate; 2′, 5′-PAP, adenosine 2′, 5′-bisphosphate; 2′:3′-cyclic PAP, adenosine 2′:3′-cyclic phosphate 5′-phosphate; AMP, adenosine 5′-monophosphate; ADP, adenosine 5′-diphosphate; pNPS, 4-nitrophenyl sulfate; pNP, 4-nitrophenol; PST, phenol sulfotransferase.

¹: To whom correspondence should be addressed at Department of Biological Science and Technology, National Chiao Tung University, 75 Po-Ai Street, Hsinchu, Taiwan, ROC. Fax: 886-3-5729288. E-mail: [email protected].

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Regular ArticleNucleotide Binding and Sulfation Catalyzed by Phenol Sulfotransferase☆

Abstract

J. Steroid Biochem. Mol. Biol.

Cell

J. Biol. Chem.

J. Mol. Biol.

Protein Express. Purif.

Anal. Biochem.

Chem. Biol. Interact.

J. Biol. Chem.

Protein Express. and Purif.

Anal. Biochem.

Methods in Enzymol.

Chem. Biol. Interact.

Regular Article
Nucleotide Binding and Sulfation Catalyzed by Phenol Sulfotransferase☆