Chiral Inversion of 1-Hydroxyethylpyrene Enantiomers Mediated by Enantioselective Sulfotransferases

doi:10.1006/bbrc.1998.8756

Biochemical and Biophysical Research Communications

Volume 247, Issue 1, 9 June 1998, Pages 181-185

https://doi.org/10.1006/bbrc.1998.8756 Get rights and content

Abstract

The benzylic alcohol 1-hydroxyethylpyrene (1-HEP) is activated to a mutagen by sulfotransferases. The sulfuric acid ester formed is difficult to detect, as it is rapidly hydrolysed back to the alcohol. Incubation of the individual enantiomers of 1-HEP with human hydroxysteroid sulfotransferase (hHST) or estrogen sulfotransferase (hEST), expressed in bacteria, led to the formation of the other enantiomer. The rates of sulfation were determined from the initial rates of chiral inversion of the alcohol, knowing that hydrolysis follows an S_N1 mechanism and therefore produces racemic alcohol. hEST showed high enantioselectivity forS-1-HEP, whereas hHST strongly preferred theR-enantiomer. The rates of sulfation of the preferred enantiomers were high, similar to those for the prototype substrates of hEST (β-estradiol) and hHST (dehydroepiandrosterone). Moreover, after a 30-min incubation ofS-1-HEP with hEST, 95% of the recovered alcohol showed theR-configuration, indicating that several cycles of sulfation and hydrolysis had led to the depletion of one enantiomer and to the enrichment of the other enantiomer.

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The human genome contains a sequence that is homologous to genes encoding soluble sulphotransferases (SULTs) based on the nucleotide sequence and possible intron/exon splice sites. The putative coding sequence (termed SULT1C3) was synthesized and integrated into a bacterial expression vector. We used the cDNA-expressed protein for raising an antiserum and studying enzyme activities. No activity was detected with 4-nitrophenol and 1-naphthol, known substrates of all other members of the human SULT1 subfamily. The activity was also negligible with paracetamol, ethanol, 5-hydroxymethylfurfural, 2-hydroxymethylpyrene, 2-(α-hydroxy)ethylpyrene, and corticosterone, compounds for which we have developed sensitive enzyme assays with direct determination of the product by HPLC-UV, HPLC-fluorescence or HPLC-MS/MS. Since diverse sulpho conjugates are chemically reactive – often short-lived and mutagenic – we expressed SULT1C3 in Ames’ Salmonella typhimurium strains TA1538 and TA100, as we had done with many other SULTs previously. The expression level of SULT1C3 protein amounted to 2% of the total cytosolic proteins, which is in the middle range of other SULTs expressed in this model. Using recombinant bacterial tester strains in mutagenicity assays, we observed SULT1C3-mediated activation of several large benzylic alcohols derived from alkylated polycyclic hydrocarbons: 1-hydroxymethylpyrene, both enantiomers of 1-(α-hydroxy)ethylpyrene, 6-hydroxymethylbenzo[a]pyrene and 6-hydroxymethylanthanthrene. 1′-Hydroxysafrole was the smallest molecule activated by SULT1C3 up to date. Our study demonstrates that SULT1C3 has sulphotransferase activity and that it prefers relatively large substrates. The substrates detected were activated to mutagens, which cannot be the regular function of the enzyme. The physiological substrates remain to be identified. Probably, they are relatively large, endogenous or common exogenous, molecules.
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Citation Excerpt :
If the sulfooxy/hydroxyl group is attached to a chiral center, the stereochemistry may be altered during this reaction cycle. Indeed, incubation of the individual enantiomers of 1-HEP with SULT in vitro led to chiral inversion [26], and (Z)-α-hydroxytamoxifen was converted to its diastereomer, (E)-α-hydroxytamoxifen, in the presence of rat hydroxysteroid sulfotransferase (rHSTa) and PAPS [73]. The chiral inversion of 1-HEP and the isotope displacement in [18O]-1-hydroxymethylpyrene have been used to determine their metabolic sulfation rates, since it is difficult to detect and quantify directly the highly reactive sulfuric acids of these alcohols.
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¹: To whom correspondence should be addressed at the German Institute of Human Nutrition (DIfE), Department of Toxicology, D-14558 Bergholz-Rehbrücke, Germany.

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Regular ArticleChiral Inversion of 1-Hydroxyethylpyrene Enantiomers Mediated by Enantioselective Sulfotransferases

Abstract

Chem.-Biol. Interact.

Chem.-Biol. Interact.

Toxicol. Lett.

J. Steroid Biochem. Mol. Biol.

Genomics

Chem.-Biol. Interact.

Mol. Cell. Endocrinol.

FASEB J.

Regular Article
Chiral Inversion of 1-Hydroxyethylpyrene Enantiomers Mediated by Enantioselective Sulfotransferases