Structure- and computational-aided engineering of an oxidase to produce isoeugenol from a lignin-derived compound

Abstract

Various 4-alkylphenols can be easily obtained through reductive catalytic fractionation of lignocellulosic biomass. Selective dehydrogenation of 4-n-propylguaiacol results in the formation of isoeugenol, a valuable flavor and fragrance molecule and versatile precursor compound. Here we present the engineering of a bacterial eugenol oxidase to catalyze this reaction. Five mutations, identified from computational predictions, are first introduced to render the enzyme more thermostable. Other mutations are then added and analyzed to enhance chemoselectivity and activity. Structural insight demonstrates that the slow catalytic activity of an otherwise promising enzyme variant is due the formation of a slowly-decaying covalent substrate-flavin cofactor adduct that can be remedied by targeted residue changes. The final engineered variant comprises eight mutations, is thermostable, displays good activity and acts as a highly chemoselective 4-n-propylguaiacol oxidase. We lastly use our engineered biocatalyst in an illustrative preparative reaction at gram-scale. Our findings show that a natural enzyme can be redesigned into a tailored biocatalyst capable of valorizing lignin-based monophenols.

Fig. 1. Conversion of 4-alkylphenols to 4-hydroxyphenyl alkenes and 4-hydroxyphenyl alcohols by vanillyl alcohol oxidase (VAO) and lignin-based synthesis of isoeugenol as fragrance, flavor or precursor of other high-value products.

The upper panel shows the mechanism by which VAO oxidizes 4-alkylphenols. The lower panel shows the catalytic pathway, which includes an engineered EUGO, by which lignin can be converted into valuable products.

Fig. 2. The difference in T_m values for FRESCO-predicted EUGO mutants.

The T_m values of 70 single mutants (2–4 replicates) determined by using the Thermofluor assay are shown with dots and the average Tm values are shown in bars. The average T_m of wild-type EUGO is 66.5 °C and set as a reference. The seven most stabilizing mutations with a T_m increase of at least 2 °C that were studied in more detail are in dark orange bars. Two of the 72 FRESCO-predicted mutants could not be expressed and were not further studied.

Fig. 3. Computational analysis for improving activity.

a The positions at the EUGO active site selected for mutagenesis (blue spheres) and the changes observed in every position according to a multiple sequence alignment (top). The FAD cofactor and the 4-n-propylguaiacol (4PG) ligand are represented as gray and light blue balls, respectively. Residues selected by the Monte Carlo algorithm for every position in the Rosetta Coupled Moves experiment are indicated (bottom). b Ligand Scores from Rosetta Coupled Moves (top) and binding energies from Autodock VINA (bottom) for all the selected variants presented in boxplots showing the median of the data as a central line, the box represents the interquartile section, which covers from 25 to 75% of the distribution, and the tips represent minimum and maximum outliers.

Fig. 4. 4-n-propylguaiacol conversion by EUGO mutants.

4-n-propylguaiacol (4PG), isoeugenol (IEUG), 4-(1-hydroxypropyl)−2-methoxyphenol (H4PG), 1-(4-hydroxy-3-methoxyphenyl)−1-propanone (HPMP) are shown in red, green, orange and blue, respectively. a 24 h 4-n-propylguaiacol conversion by cell-free extracts of EUGO single mutants; b 24 h and 72 h 4-n-propylguaiacol conversion by purified mutants of EUGO5X in order to improve chemoselectivity. Conversions of 4-n-propylguaiacol (5 mM) were carried out in the presence of enzyme (10 µM) in a 50 mM KPi buffer containing 10% v/v DMSO at pH 7.5.

Fig. 5. Structural features of S394V-EUGO5X.

a Position of V394 in the cavity, showing a sliced view of S394V-EUGO5X. The mutated V394, shown as a red sphere, is in close contact with a deep passageway buried between the two chains of the dimer. b, c Detailed views of the electron densities of 4-n-propylguaiacol bound to two S394V-EUGO5X subunits. b unbiased 2Fo-Fc electron density map showing the presence of a stretch of electron density extending from the flavin N5 atom and suggesting the presence of a covalent bond between the flavin and 4-n-propylguaiacol in subunit B. c unbiased 2Fo-Fc electron density map of a non-covalently bound ligand observed in chain A. The maps are contoured at 1.2 σ level and were calculated before the inclusion of the ligands in the refinement. In both panels, the non-covalent and covalent ligands are superimposed. Because of different crystal contacts, the crystalline protein chains might differ with regard to the rates of catalysis, substrate diffusion, or product release explaining why the covalently bound ligand accumulates preferentially in certain protein subunits. d Superposition of the covalent (in magenta) and non-covalent (in green) 4-n-propylguaiacol complexes as observed in two crystallographically independent subunits.

Fig. 6. 4-n-Propylguaiacol conversion by purified EUGO5X mutants.

4-n-propylguaiacol (4PG), isoeugenol (IEUG), 4-(1-hydroxypropyl)−2-methoxyphenol (H4PG), 1-(4-hydroxy-3-methoxyphenyl)−1-propanone (HPMP) are shown in red, green, orange and blue, respectively. Conversions of 4-n-propylguaiacol (5 mM) were carried out in the presence of enzyme (10 µM) in 50 mM KPi, 10% v/v DMSO at pH 7.5 for 3 h, except for the three indicated 1 h conversions.

Fig. 7. Structural comparison between S394V-EUGO5X (covalent and non-covalent) and PROGO.

The interactions with Y91 and Y471 are shown with dashed lines. They are involved in deprotonating 4-n-propylguaiacol (Fig. 1). The covalent and non-covalent substrates found in the structure of the S394V-EUGO5X mutant are shown in magenta and green, respectively. The substrate bound to the D151E/S394V/Q425S-EUGO5X mutant (PROGO) is shown in cyan. The side chains of the S394V-EUGO5X mutant are represented in green, with a darker green used for the subunit bearing the covalent adduct. The side chains of D151E/S394V/Q425S-EUGO5X are represented in light blue.

Fig. 8. HPLC analysis of the conversion of 4-n-propylguaiacol by PROGO.

The first two chromatograms show the elution of the reference compounds, isoeugenol (green) and 4-n-propylguaiacol (red). The chromatograms of the conversions (black) show depletion of 4-n-propylguaiacol (R_t = 9.7 min) to form isoeugenol (R_t = 8.9 min). The conversions were performed in 48 h at 25 °C using 10% (v/v) DMSO, 50 mM KPi (pH 7.5) as buffer. For the conversion by isolated enzyme, 0.5 gram (3.0 mmol) 4-n-propylguaiacol and 18.5 mg (0.30 µmol) purified PROGO was used in a 60 mL reaction volume. For the conversion by whole cells, 1.27 gram 4-n-propylguaiacol (7.6 mmol) and E. coli cells from a 150 mL culture (final OD₆₀₀ = 29) was used in a 125 mL volume.