A Xylenol Orange-Based Screening Assay for the Substrate Specificity of Flavin-Dependent para-Phenol Oxidases

Abstract

Vanillyl alcohol oxidase (VAO) and eugenol oxidase (EUGO) are flavin-dependent enzymes that catalyse the oxidation of para-substituted phenols. This makes them potentially interesting biocatalysts for the conversion of lignin-derived aromatic monomers to value-added compounds. To facilitate their biocatalytic exploitation, it is important to develop methods by which variants of the enzymes can be rapidly screened for increased activity towards substrates of interest. Here, we present the development of a screening assay for the substrate specificity of para-phenol oxidases based on the detection of hydrogen peroxide using the ferric-xylenol orange complex method. The assay was used to screen the activity of VAO and EUGO towards a set of twenty-four potential substrates. This led to the identification of 4-cyclopentylphenol as a new substrate of VAO and EUGO and 4-cyclohexylphenol as a new substrate of VAO. Screening of a small library of VAO and EUGO active-site variants for alterations in their substrate specificity led to the identification of a VAO variant (T457Q) with increased activity towards vanillyl alcohol (4-hydroxy-3-methoxybenzyl alcohol) and a EUGO variant (V436I) with increased activity towards chavicol (4-allylphenol) and 4-cyclopentylphenol. This assay provides a quick and efficient method to screen the substrate specificity of para-phenol oxidases, facilitating the enzyme engineering of known para-phenol oxidases and the evaluation of the substrate specificity of novel para-phenol oxidases.

Keywords: enzyme kinetics; flavoprotein; oxidase; screening assay; substrate specificity.

Scheme 1

Examples of reactions catalysed by vanillyl alcohol oxidase (VAO) and/or eugenol oxidase (EUGO). Both enzymes can catalyse the oxidation of vanillyl alcohol (14) to the aldehyde vanillin and of eugenol (18) to coniferyl alcohol. VAO converts 4-ethylphenol (3) to a mixture of products consisting of 4-vinylphenol and 1-(4′-hydroxyphenyl)ethanol. The hydroxylation reaction occurs enantioselectively, predominantly yielding the (R)-enantiomer of the alcohol. In contrast, previous studies failed to reveal any activity towards 3 for EUGO [13].

Figure 1

Conversion of eugenol or vanillyl alcohol by His-tagged vanillyl alcohol oxidase (His-VAO) or His-tagged eugenol oxidase (EUGO-His) followed in time using the xylenol orange assay. His-VAO ([●] for vanillyl alcohol, [♦] for eugenol) or EUGO-His ([■] for vanillyl alcohol, [▲] for eugenol) was incubated with 2 mM substrate in 50 mM potassium phosphate buffer, pH 7.5, at room temperature (19–20 °C). Reactions were allowed to proceed for the specified time, after which the amount of hydrogen peroxide formed was determined using the xylenol orange assay. Error bars represent the standard deviation of triplicate measurements. Broken lines are fits to the linear range of the data that were used to determine reaction rates (see Table 2).

Figure 2

Rate of the reactions of His-tagged vanillyl alcohol oxidase (His-VAO) and His-tagged eugenol oxidase (EUGO-His) with the compounds used in the substrate specificity screening in 50 mM potassium phosphate buffer, pH 7.5, at room temperature (19–20 °C) as determined using the xylenol orange assay. Solid bars give the reaction rates measured with the xylenol orange assay for His-VAO (green) and EUGO-His (blue). Error bars represent the standard deviation of duplicate measurements. Striped bars give k_cat values measured previously by other methods with the non-His-tagged enzymes. These k_cat values were measured under identical experimental conditions, with the exception of the temperature, which was 25 °C instead of 19–20 °C. The striped horizontal line indicates the detection limit of the xylenol orange assay, which corresponds to a reaction rate of 0.83 s⁻¹. The k_cat values were from the following sources. VAO: compounds 1, 4 [33], 5–8, 20, 21 [11], 2, 24 [14], 3, 14, 18 [13], 17 [34]. EUGO: 20, 24 [4], 19 [9], 14, 18 [13]. Numbering is according to Table 3.

Scheme 2

Oxidation of 4-cyclopentylphenol (22) to 4-(1-cyclopenten-1-yl)phenol (25) by His-tagged vanillyl alcohol oxidase (His-VAO) or His-tagged eugenol oxidase (EUGO-His), as catalysed in 50 mM potassium phosphate buffer, pH 7.5, at 25 °C.

Figure 3

Structures of the substrate-binding pockets of vanillyl alcohol oxidase (VAO) and eugenol oxidase (EUGO) with the competitive inhibitor isoeugenol bound. Structures are shown in stick representation with residues that are identical in each protein shown in grey, residues that are different in each protein shown with green (VAO) or cyan (EUGO) carbon atoms, the FAD cofactor shown with yellow carbon atoms and isoeugenol shown with magenta carbon atoms. The VAO and EUGO structures were downloaded from the PDB website (PDB identifiers: 2VAO [8] and 5FXD [9] respectively). Figure was prepared using PyMOL v. 1.3 (Schrödinger LLC, New York, NY, USA).

Figure 4

Rates of the reactions of His-tagged vanillyl alcohol oxidase (His-VAO) and His-tagged eugenol oxidase (EUGO-His) variants with the compounds used in the substrate specificity screening in 50 mM potassium phosphate buffer, pH 7.5, at room temperature (19-20 °C) as determined using the xylenol orange assay. Error bars represent the standard deviations of duplicate measurements. For all compounds that are not included in the graphs, no variants gave a signal above the detection limit of the assay. The F424G His-VAO variant was not studied, as it did not contain any flavin cofactor. For the L381W, G392F and I427T EUGO-His variants, no response higher than the detection limit was measured for any compound.