Directed aryl sulfotransferase evolution toward improved sulfation stoichiometry on the example of catechols

Ji, Yu; Islam, Shohana; Mertens, Alan M.; Sauer, Daniel F.; Dhoke, Gaurao V.; Jakob, Felix; Schwaneberg, Ulrich

doi:10.1007/s00253-019-09688-0

Biotechnologically relevant enzymes and proteins
Published: 04 March 2019

Directed aryl sulfotransferase evolution toward improved sulfation stoichiometry on the example of catechols

Yu Ji¹,
Shohana Islam^1,2,
Alan M. Mertens¹,
Daniel F. Sauer¹,
Gaurao V. Dhoke¹,
Felix Jakob^1,2 &
Ulrich Schwaneberg ORCID: orcid.org/0000-0003-2285-4202^1,2

Applied Microbiology and Biotechnology volume 103, pages 3761–3771 (2019)Cite this article

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Abstract

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 (k_cat = 41 ± 2 s⁻¹) and catalytic efficiency (k_cat/K_M = 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 k_cat value of up to 4.5-fold and sulfate transfer efficiency was also increased (up to 2.3-fold).

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Acknowledgements

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.

Funding

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].

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Lehrstuhl für Biotechnologie, RWTH Aachen University, Worringerweg 3, 52074, Aachen, Germany
Yu Ji, Shohana Islam, Alan M. Mertens, Daniel F. Sauer, Gaurao V. Dhoke, Felix Jakob & Ulrich Schwaneberg
DWI – Leibniz-Institut für Interaktive Materialien e.V., Forckenbeckstraße 50, 52056, Aachen, Germany
Shohana Islam, Felix Jakob & Ulrich Schwaneberg

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Yu Ji
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Shohana Islam
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Alan M. Mertens
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Daniel F. Sauer
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Gaurao V. Dhoke
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Felix Jakob
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Ulrich Schwaneberg
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Correspondence to Ulrich Schwaneberg.

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Ji, Y., Islam, S., Mertens, A.M. et al. Directed aryl sulfotransferase evolution toward improved sulfation stoichiometry on the example of catechols. Appl Microbiol Biotechnol 103, 3761–3771 (2019). https://doi.org/10.1007/s00253-019-09688-0

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Received: 03 December 2018
Revised: 05 February 2019
Accepted: 06 February 2019
Published: 04 March 2019
Issue Date: 02 May 2019
DOI: https://doi.org/10.1007/s00253-019-09688-0

Keywords

Directed evolution
Sulfotransferase
Catechols
4-Aminoantipyrine
p-Nitrophenyl sulfate