An Improved Spectrophotometric Method for Toluene-4-Monooxygenase Activity

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.

Keywords: HPLC; UV-Vis spectroscopy; analytical chemistry; cloning; colorimetric assay; enzyme; monooxygenase; regiospecificity.

Figure 1.. Workflow for the colorimetric quantification of naphthols.

a. Naphthalene is converted to naphthol by many monooxygenases (MOs); naphthols can then react with diazonium salts via aromatic substitution to yield brightly colored dyes with distinct spectra. b. Original workflow in which tetrazotized o-dianisidine is directly added to the aqueous medium containing naphthol prior to measurement (i). In the optimized workflow reported here (ii), the dye is extracted into ethyl acetate for improved stability. Deconvolution of UV-Vis spectra can then be used to quantify regioisomers.

Figure 2.. Dye conversion from existing and optimized workflows.

UV-Vis spectra from the published workflow for a. 1-naphthol standards and b. 2-naphthol standards. UV-Vis results from optimized workflow for c. 1-naphthol standards and d. 2-naphthol standards. Shaded error indicates sample standard deviation (n=2 for a, b; n=3 for c, d).

Figure 3.. Deconvolution of sample spectra for mixed sample quantification.

a. Representative HPLC-UV traces for regioisomer quantification (2-N is 2-naphthol and 1-N is 1-naphthol). b. Linear standard curves for HPLC in mixed samples. c. The spectra for 1-naphthol were deconvoluted using three gaussian primitives with the fit overlay and experimental data for a 20 μg/mL sample. d. Deconvoluted spectra for 2-naphthol were fit using four primitives with fit overlay and experimental data for a 20 μg/mL sample. e. All fit primitives were assessed for linearity with concentration. Regressions for the peak primitive for each species are shown (R² = 0.9995 for 1-N and R² = 0.9891 for 2-N). f. Deconvolution for a mixed sample. g. Predicted concentrations for deconvoluted mixed samples yield accurate results across the tested concentration range of 0–18 μg/mL.

Figure 4.. Biological validation of the workflow and deconvolution.

a. Crystal structure for the hydroxylase subunit of toluene-4-monooxygenase (T4MO) from Pseudomonas mendocina KR-1 [PDB: 5TDS] (32, 33). b. Magnification of the T4MO active site. Point amino acid mutations (yellow: I100, G103, A107) are known to affect naphthalene oxidation regiospecificity (11, 32, 33). c. Workflow for T4MO expression and naphthalene exposure in E. coli. d. Regiospecificity determination using HPLC and colorimetric readout for cultures induced with IPTG at varying concentrations. Error bars represent the standard deviation for n = 3 replicates.