Directed aryl sulfotransferase evolution toward improved sulfation stoichiometry on the example of catechols
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Sulfation is an important way for detoxifying xenobiotics and endobiotics including catechols. Enzymatic sulfation occurs usually with high chemo- and/or regioselectivity under mild reaction conditions. In this study, a two-step p-NPS-4-AAP screening system for laboratory evolution of aryl sulfotransferase B (ASTB) was developed in 96-well microtiter plates to improve the sulfate transfer efficiency toward catechols. Increased transfer efficiency and improved sulfation stoichiometry are achieved through the two-step screening procedure in a one-pot reaction. In the first step, the p-NPS assay is used (detection of the colorimetric by-product, p-nitrophenol) to determine the apparent ASTB activity. The sulfated product, 3-chlorocatechol-1-monosulfate, is quantified by the 4-aminoantipyrine (4-AAP) assay in the second step. Comparison of product formation to p-NPS consumption ensures successful directed evolution campaigns of ASTB. Optimization yielded a coefficient of variation below 15% for the two-step screening system (p-NPS-4-AAP). In total, 1760 clones from an ASTB-SeSaM library were screened toward the improved sulfation activity of 3-chlorocatechol. The turnover number (kcat = 41 ± 2 s−1) and catalytic efficiency (kcat/KM = 0.41 μM−1 s−1) of the final variant ASTB-M5 were improved 2.4- and 2.3-fold compared with ASTB-WT. HPLC analysis confirmed the improved sulfate stoichiometry of ASTB-M5 with a conversion of 58% (ASTB-WT 29%; two–fold improvement). Mass spectrometry (MS) and nuclear magnetic resonance spectroscopy (NMR) confirmed the chemo- and regioselectivity, which yielded exclusively 3-chlorocatechol-1-monosulfate. For all five additionally investigated catechols, the variant ASTB-M5 achieved an improved kcat value of up to 4.5-fold and sulfate transfer efficiency was also increased (up to 2.3-fold).
KeywordsDirected evolution Sulfotransferase Catechols 4-Aminoantipyrine p-Nitrophenyl sulfate
The authors thank Dr. Kilian E. C. Smith and David Kämpfer for helping with the MS measurement and Prof. Dr. Jun Okuda for the NMR support.
This research was funded by the China Scholarship Council (CSC) (No. 201608080082) and German Federal Ministry of Education and Research (BMBF) under the projects “FuPol” (Functionalization of Polymers [FKZ: 031A227F]) alliance and IBÖ-03: BioSulfa-Effiziente Sulfatierung von Biomolekülen [FKZ: 031B0255].
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
This article does not provide any research involving human participants and/or animal experiments.
- Ahmadi S, Shadman M, Hossini H, Hashemi S (2016) Catechol removal using MWCNTs from synthetic solutions: modeling, equilibrium and kinetics. J Mater Environ Sci 7(10):3885–3894Google Scholar
- Bukowska B, Marczak A, MichaŃowicz J, Wisniewska K (2009) Effects of phenol, catechol, chloro-and metylphenol on human erythrocyte membrane (in vitro). Pol J Environ Stud 18(4):569–577Google Scholar
- Capasso R, Evidente A, Schivo L, Orru G, Marcialis M, Cristinzio G (1995) Antibacterial polyphenols from olive oil mill waste waters. J Appl Bacteriol 79(4):393–398. https://doi.org/10.1111/j.1365-2672.1995.tb03153.x Google Scholar
- Fang C, Kim H, Barnes RC, Talcott ST, Mertens-Talcott SU (2018) Obesity-associated diseases biomarkers are differently modulated in lean and obese individuals and inversely correlated to plasma polyphenolic metabolites after 6 weeks of mango (Mangifera Indica L.) Consumption. Mol Nutr Food Res 62(14):1800129. https://doi.org/10.1002/mnfr.201800129 Google Scholar
- Gillam EM, Hayes MA (2013) The evolution of cytochrome P450 enzymes as biocatalysts in drug discovery and development. Curr Top Med Chem 13(18):2254–2280Google Scholar
- Olson JA, Adler-Moore JP, Smith P, Proffitt RT (2005) Treatment of Candida glabrata infection in immunosuppressed mice by using a combination of liposomal amphotericin B with caspofungin or micafungin. Antimicrob Agents Chemother 49(12):4895–4902. https://doi.org/10.1128/AAC.49.12.4895-4902.2005
- Pimpão RC, Dew T, Figueira ME, McDougall GJ, Stewart D, Ferreira RB, Santos CN, Williamson G (2014) Urinary metabolite profiling identifies novel colonic metabolites and conjugates of phenolics in healthy volunteers. Mol Nutr Food Res 58(7):1414–1425. https://doi.org/10.1002/mnfr.201300822 Google Scholar
- Powell KA, Ramer SW, del Cardayré SB, Stemmer WP, Tobin MB, Longchamp PF, Huisman GW (2001) Directed evolution and biocatalysis. Angew Chem Int Ed 40(21):3948–3959. https://doi.org/10.1002/1521-3773(20011105)40:21<3948::AID-ANIE3948>3.0.CO;2-N Google Scholar
- Rivera-Marrero CA, Ritzenthaler JD, Newburn SA, Roman J, Cummings RD (2002) Molecular cloning and expression of a novel glycolipid sulfotransferase in Mycobacterium tuberculosis. Microbiology 148(3):783–792. https://doi.org/10.1099/00221287-148-3-783
- Roubalová L, Purchartová K, Papoušková B, Vacek J, Křen V, Ulrichová J, Vrba J (2015) Sulfation modulates the cell uptake, antiradical activity and biological effects of flavonoids in vitro: an examination of quercetin, isoquercitrin and taxifolin. Biorg Med Chem 23(17):5402–5409. https://doi.org/10.1016/j.bmc.2015.07.055 Google Scholar
- Sperry S, Crews P (1997) Haliclostanone sulfate and halistanol sulfate from an Indo-Pacific Haliclona sponge. J Nat Prod 60(1):29–32. https://doi.org/10.1021/np960592s
- van der Horst MA, van Lieshout JF, Bury A, Hartog AF, Wever R (2012) Sulfation of various alcoholic groups by an arylsulfate sulfotransferase from Desulfitobacterium hafniense and synthesis of estradiol sulfate. Adv Synth Catal 354(18):3501–3508. https://doi.org/10.1002/adsc.201200564