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ORIGINAL RESEARCH article

Front. Synth. Biol.
Sec. Ecosystems and Biodiversity Sustainability
Volume 2 - 2024 | doi: 10.3389/fsybi.2024.1419557

Global incorporation of meta-fluorotyrosine or meta-fluorophenylalanine into 1,2-catechol dioxygenase modulates substrate binding affinities

Provisionally accepted
Michael C. Wong Michael C. Wong 1Thomas E. Grant Thomas E. Grant 2,3Hamid Reza Karbalaei-Heidari Hamid Reza Karbalaei-Heidari 1Anna C. Robotham Anna C. Robotham 2Matthew E. Loewen Matthew E. Loewen 4Antony St-Jacques Antony St-Jacques 2Ned Budisa Ned Budisa 1Michele C. Loewen Michele C. Loewen 2,3*
  • 1 University of Manitoba, Winnipeg, Manitoba, Canada
  • 2 National Research Council Canada (NRC), Ottawa, Canada
  • 3 University of Ottawa, Ottawa, Ontario, Canada
  • 4 University of Saskatchewan, Saskatoon, Saskatchewan, Canada

The final, formatted version of the article will be published soon.

    Previous studies have shown how replacing canonical residues with isosteric amino acid analogs in enzymes can predictably alter biocatalytic activities by introducing variations in the first and second shell residues relative to the binding pocket. In this study, we explore the global integration of amino acid analogs into 1,2-catechol dioxygenase from Rhodococcus opacus (Rho 1,2-CTD), an industrial enzyme containing non-heme iron that is vital for phenol ring degradation. Methods: We replaced tyrosine residues in the first shell of the binding pocket, phenylalanine residues in the second shell, and methionine residues near the binding pocket with m-fluorotyrosine (m-FY), m-fluorophenylalanine (m-FF), and Nle, respectively, using auxotrophic Escherichia coli strains. Results: The expression, purification and incorporation of m-FY and m-FF into the Rho 1,2-CTD was successful, while the Nleinsertion did not work. The structural characterization of the resulting m-FF-and m-FYcontaining variants provided a mechanistic framework and a plausible explanation for the results of the kinetic analyses of the native enzyme and fluorinated variants. Discussion: Our findings demonstrate the impact of fluorination on the activity of 1,2-catechol dioxygenase, revealing its influence on residues near the substrate (first shell) as well as those distant from the binding pocket (second shell). This provides a robust foundation for future activity engineering efforts.

    Keywords: global incorporation, Non-canonical amino acid, meta-fluorotyrosine, Norleucine, 1,2-catechol dioxygenase, Substrate affinity, meta-fluorophenylalanine

    Received: 18 Apr 2024; Accepted: 21 Jun 2024.

    Copyright: © 2024 Wong, Grant, Karbalaei-Heidari, Robotham, Loewen, St-Jacques, Budisa and Loewen. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

    * Correspondence: Michele C. Loewen, National Research Council Canada (NRC), Ottawa, Canada

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