The preparation of pure metabolites of bioactive compounds, particularly (poly)phenols, is essential for the accurate determination of their pharmacological profiles in vivo. Since the extraction of these metabolites from biological material is tedious and impractical, they can be synthesized enzymatically in vitro by bacterial PAPS-independent aryl sulfotransferases (ASTs). However, only a few ASTs have been studied and used for (poly)phenol sulfation. This study introduces new fully characterized recombinant ASTs selected according to their similarity to the previously characterized ASTs. These enzymes, produced in Escherichia coli, were purified, biochemically characterized, and screened for the sulfation of nine flavonoids and two phenolic acids using p-nitrophenyl sulfate. All tested compounds were proved to be substrates for the new ASTs, with kaempferol and luteolin being the best converted acceptors. ASTs from Desulfofalx alkaliphile (DalAST) and Campylobacter fetus (CfAST) showed the highest efficiency in the sulfation of tested polyphenols. To demonstrate the efficiency of the present sulfation approach, a series of new authentic metabolite standards, regioisomers of kaempferol sulfate, were enzymatically produced, isolated, and structurally characterized.

Department of Biochemistry and Microbiology University of Chemistry and Technology Prague Technická 3 Prague 6 CZ 166 28 Czech Republic

Department of Genetics and Microbiology Faculty of Science Charles University Albertov 6 Prague 2 CZ 128 43 Czech Republic

Institute of Microbiology of the Czech Academy of Sciences Vídeňská 1083 Prague 4 CZ 142 00 Czech Republic

Zobrazit více v PubMed

Teles Y. C. F.; Souza M. S. R.; Souza M. Sulphated flavonoids: biosynthesis, structures, and biological activities. Molecules 2018, 23 (2), 480.10.3390/molecules23020480. PubMed DOI PMC

Kerimi A.; Williamson G. At the interface of antioxidant signalling and cellular function: Key polyphenol effects. Mol. Nutr. Food Res. 2016, 60 (8), 1770–1788. 10.1002/mnfr.201500940. PubMed DOI PMC

Sak K.Anticancer Action of Sulfated Flavonoids as Phase II Metabolites. In Food Bioconversion; Grumezescu A. M., Holban A. M., Eds.; Academic Press Ltd-Elsevier Science Ltd, 2017; Vol. 2, pp 207–236

Chapman E.; Best M. D.; Hanson S. R.; Wong C. H. Sulfotransferases: Structure, mechanism, biological activity, inhibition, and synthetic utility. Angew. Chem., Int. Ed. 2004, 43 (27), 3526–3548. 10.1002/anie.200300631. PubMed DOI

Roubalová L.; Purchartová K.; Papoušková B.; Vacek J.; Křen V.; Ulrichová J.; Vrba J. Sulfation modulates the cell uptake, antiradical activity and biological effects of flavonoids in vitro: An examination of quercetin, isoquercitrin and taxifolin. Bioorg. Med. Chem. 2015, 23 (17), 5402–5409. 10.1016/j.bmc.2015.07.055. PubMed DOI

Leonardo C. C.; Doré S. Dietary flavonoids are neuroprotective through Nrf2-coordinated induction of endogenous cytoprotective proteins. Nutr. Neurosci. 2011, 14 (5), 226–236. 10.1179/1476830511Y.0000000013. PubMed DOI PMC

Saha S.; Hollands W.; Needs P. W.; Ostertag L. M.; de Roos B.; Duthie G. G.; Kroon P. A. Human O-sulfated metabolites of (−)-epicatechin and methyl-(−)-epicatechin are poor substrates for commercial aryl-sulfatases: Implications for studies concerned with quantifying epicatechin bioavailability. Pharmacol. Res. 2012, 65 (6), 592–602. 10.1016/j.phrs.2012.02.005. PubMed DOI

Mueller J. W.; Gilligan L. C.; Idkowiak J.; Arlt W.; Foster P. A. The regulation of steroid action by sulfation and desulfation. Endocr. Rev. 2015, 36 (5), 526–563. 10.1210/er.2015-1036. PubMed DOI PMC

Almeida A. F.; Borge G. I. A.; Piskula M.; Tudose A.; Tudoreanu L.; Valentová K.; Williamson G.; Santos C. N. Bioavailability of quercetin in humans with a focus on interindividual variation. Compr. Rev. Food. Sci. Food Saf. 2018, 17 (3), 714–731. 10.1111/1541-4337.12342. PubMed DOI

Hemmerich S.; Verdugo D.; Rath V. L. Strategies for drug discovery by targeting sulfation pathways. Drug Discovery Today 2004, 9 (22), 967–975. 10.1016/S1359-6446(04)03261-1. PubMed DOI

Correia-da-Silva M.; Sousa E.; Pinto M. M. M. Emerging sulfated flavonoids and other polyphenols as drugs: nature as an inspiration. Med. Res. Rev. 2014, 34 (2), 223–279. 10.1002/med.21282. PubMed DOI

Al-Horani R. A.; Desai U. R. Chemical sulfation of small molecules-advances and challenges. Tetrahedron 2010, 66 (16), 2907–2918. 10.1016/j.tet.2010.02.015. PubMed DOI PMC

van der Horst M. A.; van Lieshout J. F. T.; Bury A.; Hartog A. F.; Wever R. Sulfation of various alcoholic groups by an arylsulfate sulfotransferase from Desulfitobacterium hafniense and synthesis of estradiol sulfate. Adv. Synth. Catal. 2012, 354 (18), 3501–3508. 10.1002/adsc.201200564. DOI

Malojčić G.; Owen R. L.; Glockshuber R. Structural and mechanistic insights into the PAPS-independent sulfotransfer catalyzed by bacterial aryl sulfotransferase and the role of the DsbL/DsbI system in its folding. Biochemistry 2014, 53 (11), 1870–1877. 10.1021/bi401725j. PubMed DOI

Islam S.; Laaf D.; Infanzón B.; Pelantová H.; Davari M. D.; Jakob F.; Křen V.; Elling L.; Schwaneberg U. Knowvolution campaign of an aryl sulfotransferase increases activity toward cellobiose. Chem.—Eur. J. 2018, 24 (64), 17117–17124. 10.1002/chem.201803729. PubMed DOI

Brodsky K.; Káňová K.; Konvalinková D.; Slámová K.; Pelantová H.; Valentová K.; Bojarová P.; Křen V.; Petrásková L. Bacterial aryl sulfotransferases in selective and sustainable sulfation of biologically active compounds using novel sulfate donors. ChemSusChem 2022, 15 (18), 9.10.1002/cssc.202201253. PubMed DOI

Valentová K.; Káňová K.; Di Meo F.; Pelantová H.; Chambers C.; Rydlová L.; Petrásková L.; Křenková A.; Cvačka J.; Trouillas P.; Křen V. Chemoenzymatic preparation and biophysical properties of sulfated quercetin metabolites. Int. J. Mol. Sci. 2017, 18 (11), 2231.10.3390/ijms18112231. PubMed DOI PMC

Yun H. J.; Kwon A. R.; Choi E. C. Bacterial arylsulfate sulfotransferase as a reporter system. Microbiol. Immunol. 2001, 45 (10), 673–678. 10.1111/j.1348-0421.2001.tb01301.x. PubMed DOI

Grimshaw J. P. A.; Stirnimann C. U.; Brozzo M. S.; Malojcic G.; Grütter M. G.; Capitani G.; Glockshuber R. DsbL and DsbI form a specific dithiol oxidase system for periplasmic arylsulfate sulfotransferase in uropathogenic Escherichia coli. J. Mol. Biol. 2008, 380 (4), 667–680. 10.1016/j.jmb.2008.05.031. PubMed DOI

Malojčić G.; Glockshuber R. The PAPS-independent aryl sulfotransferase and the alternative disulfide bond formation system in pathogenic bacteria. Antioxid. Redox Signal. 2010, 13 (8), 1247–1259. 10.1089/ars.2010.3119. PubMed DOI

Kim D. H.; Konishi L.; Kobashi K. Purification, characterization and reaction-mechanism of novel arylsulfotransferase obtained from an anaerobic bacterium of human intestine. Biochim. Biophys. Acta 1986, 872 (1–2), 33–41. 10.1016/0167-4838(86)90144-5. PubMed DOI

Malojčić G.; Owen R. L.; Grimshaw J. P. A.; Brozzo M. S.; Dreher-Teo H.; Glockshuber R. A structural and biochemical basis for PAPS-independent sulfuryl transfer by aryl sulfotransferase from uropathogenic Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 2008, 105 (49), 19217–19222. 10.1073/pnas.0806997105. PubMed DOI PMC

Purchartová K.; Valentová K.; Pelantová H.; Marhol P.; Cvačka J.; Havlíček L.; Křenková A.; Vavříková E.; Biedermann D.; Chambers C. S.; Křen V. Prokaryotic and eukaryotic aryl sulfotransferases: sulfation of quercetin and its derivatives. ChemCatChem 2015, 7 (19), 3152–3162. 10.1002/cctc.201500298. DOI

Káňová K.; Petrásková L.; Pelantová H.; Rybková Z.; Malachová K.; Cvačka J.; Křen V.; Valentová K. Sulfated metabolites of luteolin, myricetin, and ampelopsin: chemoenzymatic preparation and biophysical properties. J. Agric. Food Chem. 2020, 68 (40), 11197–11206. 10.1021/acs.jafc.0c03997. PubMed DOI

Carregosa D.; Carecho R.; Figueira I.; N Santos C. Low-molecular weight metabolites from polyphenols as effectors for attenuating neuroinflammation. J. Agric. Food Chem. 2020, 68 (7), 1790–1807. 10.1021/acs.jafc.9b02155. PubMed DOI

Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 1976, 72 (1–2), 248–254. 10.1016/0003-2697(76)90527-3. PubMed DOI

Marhol P.; Hartog A. F.; van der Horst M. A.; Wever R.; Purchartová K.; Fuksová K.; Kuzma M.; Cvačka J.; Křen V. Preparation of silybin and isosilybin sulfates by sulfotransferase from Desulfitobacterium hafniense. J. Mol. Catal. B-Enzym. 2013, 89, 24–27. 10.1016/j.molcatb.2012.12.005. DOI

Valentová K.; Purchartová K.; Rydlová L.; Roubalová L.; Biedermann D.; Petrásková L.; Křenková A.; Pelantová H.; Holečková-Moravcová V.; Tesařová E.; Cvačka J.; Vrba J.; Ulrichová J.; Křen V. Sulfated metabolites of flavonolignans and 2,3-dehydroflavonolignans: preparation and properties. Int. J. Mol. Sci. 2018, 19 (8), 2349.10.3390/ijms19082349. PubMed DOI PMC

Kaci H.; Bodnárová S.; Fliszár-Nyúl E.; Lemli B.; Pelantová H.; Valentová K.; Bakos É.; Özvegy-Laczka C.; Poór M. Interaction of luteolin, naringenin, and their sulfate and glucuronide conjugates with human serum albumin, cytochrome P450 (CYP2C9, CYP2C19, and CYP3A4) enzymes and organic anion transporting polypeptide (OATP1B1 and OATP2B1) transporters. Biomed. Pharmacother. 2023, 157, 114078.10.1016/j.biopha.2022.114078. PubMed DOI

Kim D. H.; Kim B.; Kim H. S.; Sohng I. S.; Kobashi K. Sulfation of parabens and tyrosylpeptides by bacterial arylsulfate sulfotransferases. Biol. Pharm. Bull. 1994, 17 (10), 1326–1328. 10.1248/bpb.17.1326. PubMed DOI

Ayuso-Fernández I.; Galmés M. A.; Bastida A.; García-Junceda E. Aryl sulfotransferase from Haliangium ochraceum: a versatile tool for the sulfation of small molecules. ChemCatChem 2014, 6 (4), 1059–1065. 10.1002/cctc.201300853. DOI

Hartog A. F.; Wever R. Substrate engineering and its synthetic utility in the sulfation of primary aliphatic alcohol groups by a bacterial aryl sulfotransferase. Adv. Synth. Catal. 2015, 357 (12), 2629–2632. 10.1002/adsc.201500482. DOI

Hartog A. F.; Wever R. Sulfation made easy: a new versatile donor for enzymatic sulfation by a bacterial aryl sulfotransferase. J. Mol. Catal. B-Enzym. 2016, 129, 43–46. 10.1016/j.molcatb.2016.04.007. DOI

Talley K.; Alexov E. On the pH-optimum of activity and stability of proteins. Proteins 2010, 78 (12), 2699–2706. 10.1002/prot.22786. PubMed DOI PMC

Rajhard S.; Hladnik L.; Vicente F. A.; Srčič S.; Grilc M.; Likozar B. Solubility of luteolin and other polyphenolic compounds in water, nonpolar, polar aprotic and protic solvents by applying FTIR/HPLC. Processes 2021, 9 (11), 1952.10.3390/pr9111952. DOI

Kobashi K.; Kim D. H.; Morikawa T. A novel type of aryl sulfotransferase. J. Protein Chem. 1987, 6 (3), 237–244. 10.1007/BF00250287. DOI

van der Horst M. A.; Hartog A. F.; El Morabet R.; Marais A.; Kircz M.; Wever R. Enzymatic sulfation of phenolic hydroxy groups of various plant metabolites by an arylsulfotransferase. Eur. J. Org Chem. 2015, 2015 (3), 534–541. 10.1002/ejoc.201402875. DOI

Kolaříková V.; Brodsky K.; Petrásková L.; Pelantová H.; Cvačka J.; Havlíček L.; Křen V.; Valentová K. Sulfation of phenolic acids: chemoenzymatic vs. chemical synthesis. Int. J. Mol. Sci. 2022, 23 (23), 15171.10.3390/ijms232315171. PubMed DOI PMC