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Engineering P450 Monooxygenases for Highly Regioselective and Active p-Hydroxylation of m-Alkylphenols
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    Engineering P450 Monooxygenases for Highly Regioselective and Active p-Hydroxylation of m-Alkylphenols
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    • Ren-Jie Li
      Ren-Jie Li
      Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, Singapore 117585, Singapore
      Synthetic Biology for Clinical and Technological Innovation (SynCTI), National University of Singapore, 28 Medical Drive, Singapore 117456, Singapore
      More by Ren-Jie Li
    • Kaiyuan Tian
      Kaiyuan Tian
      Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, Singapore 117585, Singapore
      More by Kaiyuan Tian
    • Xirui Li
      Xirui Li
      Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, Singapore 117585, Singapore
      More by Xirui Li
    • Anand Raghavendra Gaikaiwari
      Anand Raghavendra Gaikaiwari
      Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, Singapore 117585, Singapore
    • Zhi Li*
      Zhi Li
      Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, Singapore 117585, Singapore
      Synthetic Biology for Clinical and Technological Innovation (SynCTI), National University of Singapore, 28 Medical Drive, Singapore 117456, Singapore
      *Email: [email protected]
      More by Zhi Li
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    ACS Catalysis

    Cite this: ACS Catal. 2022, 12, 10, 5939–5948
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    https://doi.org/10.1021/acscatal.1c06011
    Published May 5, 2022
    Copyright © 2022 American Chemical Society

    Abstract

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    Regioselective hydroxylations of aromatic compounds are useful reactions but often lack appropriate catalysts. Here a group of P450BM3 mutants (R47I/A82F/A328F, R47L/Y51F/F87V/L188P/I401P, R47I/Y51F/F87V, R47L/Y51F/F87V/L181Q/L188P/I401P, and R47I/F87V/L188P) were developed as unique catalysts for the p-hydroxylation of m-alkylphenols 1ae with high regioselectivity (91–99%) and conversion (95–99%) to produce the corresponding useful and valuable m-alkylbenzene-1,4-diols 2ae, respectively. The mutated hydroxylases were developed by protein engineering of P450BM3 monooxygenase via site-directed mutagenesis based on designed mutations to reshape the substrate binding pocket and access channel. Several engineered P450BM3 mutants showed good catalytic efficiency (kcat/KM of 234–381 mM–1 min–1) for the p-hydroxylations of m-alkylphenols 1ae, respectively. Molecular docking and simulation gave some insights into the structure-based understanding of the enhanced regioselectivity and activity for the developed P450BM3 mutants, including the shorter distance between heme-oxygen atom and C4-carbon (p-position) of substrates than the wild-type enzyme in the catalytic pockets. Preparative biohydroxylations of m-alkylphenols 1ae were demonstrated by using E. coli cells coexpressing individual P450BM3 mutants and glucose dehydrogenase GDH, giving high-yielding synthesis of useful and valuable m-alkylbenzene-1,4-diols 2ae.

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    • Chemicals, biochemicals, strains and primers; analytic methods; molecular dynamics simulation; GC-MS chromatograms; and NMR spectra (PDF)

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    ACS Catalysis

    Cite this: ACS Catal. 2022, 12, 10, 5939–5948
    Click to copy citationCitation copied!
    https://doi.org/10.1021/acscatal.1c06011
    Published May 5, 2022
    Copyright © 2022 American Chemical Society

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