Selective Oxidations Using a Cytochrome P450 Enzyme Variant Driven with Surrogate Oxygen Donors and Light

Abstract

Cytochrome P450 monooxygenase enzymes are versatile catalysts, which have been adapted for multiple applications in chemical synthesis. Mutation of a highly conserved active site threonine to a glutamate can convert these enzymes into peroxygenases that utilise hydrogen peroxide (H₂ O₂ ). Here, we use the T252E-CYP199A4 variant to study peroxide-driven oxidation activity by using H₂ O₂ and urea-hydrogen peroxide (UHP). We demonstrate that the T252E variant has a higher stability to H₂ O₂ in the presence of substrate that can undergo carbon-hydrogen abstraction. This peroxygenase variant could efficiently catalyse O-demethylation and an enantioselective epoxidation reaction (94 % ee). Neither the monooxygenase nor peroxygenase pathways of the P450 demonstrated a significant kinetic isotope effect (KIE) for the oxidation of deuterated substrates. These new peroxygenase variants offer the possibility of simpler cytochrome P450 systems for selective oxidations. To demonstrate this, a light driven H₂ O₂ generating system was used to support efficient product formation with this peroxygenase enzyme.

Keywords: biocatalysis; heme enzymes; monooxygenases; peroxygenases; protein engineering.

Scheme 1

Catalytic cycle of P450 monooxygenases. The peroxide shunt pathway which enables the enzyme to bypass the electron transfer steps is highlighted.

Figure 1

Rate of heme bleaching of the T252E‐CYP199A4 enzyme (3 μM) exposed to 60 mM H₂O₂ with (a) 1 mM 4‐methoxybenzoic acid, (b) in the absence of substrate and (c) 1 mM 4‐trifluoromethoxybenzoic acid. After 1 minute spectra were recorded at 2 minute intervals.

Figure 2

Crystal structure of 4‐trifluoromethoxybenzoic acid bound to T252E‐CYP199A5 solved at 1.95 Å. A composite omit map of the 4‐trifluoromethoxybenzoic acid substrate is shown as a grey mesh contoured at 1.0 (1.5 Å carve). b) The superimposed crystal structures of T252E‐CYP199A4 bound to 4‐trifluoromethoxybenzoic acid (yellow) and 4‐methoxybenzoic acid (green). When bound to the fluorinated substrate, the F298 residue moves away from the heme centre compared to 4‐methoxybenzoic acid to accommodate the trifluoro moiety.

Scheme 2

Oxidation of para‐substituted benzoic acids carried out by T252E‐CYP199A4.

Figure 3

(a) HPLC analysis of 4‐methoxybenzoic acid O‐demethylation by T252E‐CYP199A4 (1 μM) using different concentrations of H₂O₂. Products are labelled as is the internal standard (IS). (b) A comparison of the total turnover numbers (TTN) obtained with T252E‐CYP199A4 (5 μM or 3 μM) in the presence of 1 mM 4‐methoxybenzoic acid and different concentrations of UHP and H₂O₂ (32 mM or 64 mM). Reactions were conducted at 30 °C and left for 24 h.

Figure 4

(a) Spin state shift analysis of 4‐(methoxy‐d₃) benzoic‐d₄ acid with T252E‐CYP199A4. (b) HPLC analysis of 4‐methoxybenzoic acid (black) and 4‐(methoxy‐d₃) benzoic‐d₄ acid (red) with T252E‐CP199A4 in the presence of 1 mM H₂O₂ after 24 h of incubation at 16 °C and 70 RPM. Substrate concentration used was 100 μM.

Figure 5

(a) Time course of 4‐vinylbenzoic acid conversion into the corresponding epoxide by peroxygenase activity of WT and T252E CYP199A4 isoforms (b) Enantioselective HPLC analysis of the epoxide generated by the T252E mutant in a H₂O₂‐driven reaction (60 mM H₂O₂) over a 60‐minute period (black). In purple is a control reaction omitting the P450.

Figure 6

GC‐MS Analysis of light driven oxidation of tetradecanoic acid (C14) by CYP152A1 (black, P450BSβ). In red is a control reaction with 10 mM H₂O₂ and no P450 added. Conditions used are 5 μM P450, 200 μM FMN, 1 mM EDTA and 100 μM substrate for the light‐driven reaction. Other metabolites were indicated by the length of the fatty acid chain (i. e., C10 for decanoic acid) and by α or β hydroxylation (i. e., α‐C14‐OH for α‐hydroxy tetradecanoic acid).

Scheme 3

Reaction scheme for the hydroxylation of fatty acids by P450Bsβ and subsequent α‐keto decarboxylation.

Figure 7

(a) Light driven turnovers of T252E‐CYP199A4 (1 μM) and substrate (200 μM) with a control reaction in the dark. (b) Time course experiments of light activated T252E‐CYP199A4 reaction with 4‐methoxybenzoic acid. Conditions used are CYP199A4‐T252E (3 μM), FMN (200 μM), EDTA (1 mM) and 4‐methoxyBA (200 μM). (c) Optimisation of light‐activated systems were then carried out with different enzyme concentrations (1 μM, 3 μM and 5 μM) with 200 μM FMN Reaction time was 20 hr at room temperature).

Scheme 4

Synthesis of 4‐(methoxy‐d₃)benzoic‐d₄ acid. Reagents and conditions: (a) Pd/C, NaOH, D₂O, 210 °C, 2 h, 4 %, 89.6 %D; (b) i) CD₃I, K₂CO₃, reflux, 10 h, ii) LiOH (aq.), MeOD, THF, rt, 16 h, 62 %, 93.3 %D.