An Improved Spectrophotometric Method for Toluene-4-Monooxygenase Activity.

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  • Additional Information
    • Source:
      Publisher: Wiley-VCH Country of Publication: Germany NLM ID: 9513783 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1521-3765 (Electronic) Linking ISSN: 09476539 NLM ISO Abbreviation: Chemistry Subsets: MEDLINE
    • Publication Information:
      Original Publication: Weinheim, Germany : Wiley-VCH
    • Subject Terms:
      Oxygenases*/chemistry ; Mixed Function Oxygenases*
    • Abstract:
      Monooxygenases, an important class of enzymes, have been the subject of enzyme engineering due to their high activity and versatile substrate scope. Reactions performed by these biocatalysts have long been monitored by a colorimetric method involving the coupling of a dye precursor to naphthalene hydroxylation products generated by the enzyme. Despite the popularity of this method, we found the dye product to be unstable, preventing quantitative readout. By incorporating an extraction step to solubilize the dye produced, we have improved this assay to the point where quantitation of enzyme activity is possible. Further, by incorporating spectral deconvolution, we have, for the first time, enabled independent quantification of the two possible regioisomeric products: 1-naphthol and 2-naphthol. Previously, such analysis was only possible with chromatographic separation, increasing the cost and complexity of analysis. The efficacy of our improved workflow was evaluated by monitoring the activity of a toluene-4-monooxygenase enzyme from Pseudomonas mendocina KR-1. Our colorimetric regioisomer quantification was found to be consistent with chromatographic analysis by HPLC. The development and validation of a quantitative colorimetric assay for monooxygenase activity that enables regioisomeric distinction and quantification represents a significant advance in analytical methods to monitor enzyme activity. By maintaining facile, low-cost, high-throughput readout while incorporating quantification, this assay represents an important alternative to more expensive chromatographic quantification techniques.
      (© 2023 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH.)
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    • Grant Information:
      P30 ES002109 United States ES NIEHS NIH HHS; P42 ES027707 United States ES NIEHS NIH HHS; P30-ES002109 United States ES NIEHS NIH HHS
    • Contributed Indexing:
      Keywords: HPLC; UV-Vis spectroscopy; analytical chemistry; cloning; colorimetric assay; enzyme; monooxygenase; regiospecificity
    • Accession Number:
      EC 1.14.13.- (toluene-4-monooxygenase)
      EC 1.13.- (Oxygenases)
      EC 1.- (Mixed Function Oxygenases)
    • Publication Date:
      Date Created: 20230102 Date Completed: 20230404 Latest Revision: 20240923
    • Publication Date:
      20240923
    • Accession Number:
      PMC10423644
    • Accession Number:
      10.1002/chem.202203322
    • Accession Number:
      36593585