Spectroscopic studies of the interaction between phosphorus heterocycles and cytochrome P450

Abstract

P450 fatty acid decarboxylase OleT from Staphylococcus aureus (OleT_SA) is a novel cytochrome P450 enzyme that catalyzes the oxidative decarboxylation of fatty acids to yield primarily terminal alkenes and CO₂ or minor α- and β-hydroxylated fatty acids as side-products. In this work, the interactions between a series of cycloalkyl phosphorus heterocycles (CPHs) and OleT_SA were investigated in detail by fluorescence titration experiment, ultraviolet-visible (UV-vis) and ³¹P NMR spectroscopies. Fluorescence titration experiment results clearly showed that a dynamic quenching occurred when CPH-6, a representative CPHs, interacted with OleT_SA with a binding constant value of 15.2 × 10⁴ M^-1 at 293 K. The thermodynamic parameters (ΔH, ΔS and ΔG) showed that the hydrogen bond and van der Waals force played major roles in the interaction between OleT_SA and CPHs. The UV-vis and ³¹P NMR studies indicated the penetration of CPH-6 into the interior environment of OleT_SA, which greatly affects the enzymatic activity of OleT_SA. Therefore, our study revealed an effective way to use phosphorus heterocyclic compounds to modulate the activity of cytochrome P450 enzymes.

Keywords: Cytochrome P450; Interaction; OleT; Phosphorus heterocycles; Spectroscopy.

Graphical abstract

Fig. 1

Structure of representative phosphorus heterocycles as novel pharmaceuticals.

Fig. 2

Synthesis of cycloalkyl phosphorus heterocycles (CPHs).

Fig. 3

The changes in the intrinsic fluorescence intensity of OleT_SA measured at the excitation wavelength (λ_em) of 280 nm in the presence of different concentrations of CPHs (0, 5, 10, 15, 20, 25, and 30 μM) at 298 K.

Fig. 4

(A): Fluorescence spectra of CPH-6 measured at the excitation wavelength (λ_em) of 250 nm alongside with various concentrations of OleT_SA (0, 0.2, 0.6, 2, 4, 6, 8, and 10 μM) at 298 K. (B): Stern-Volmer plot for binding OleT_SA with CPH-6 at 293, 298, and 303 K, respectively.

Fig. 5

Double logarithmic plots for OleT_SA-CPH-6 complexes.

Fig. 6

The Van't Hoff plot for the binding of OleT_SA with CPH-6.

Fig. 7

UV–Vis binding titrations of CPH-6 with OleT_SA. 10 μM OleT_SA was used.

Fig. 8

³¹P NMR spectra of CPH-6-OleT_SA, CPH-6-BSA, and CPH-6 only. 50 μM of OleT_SA or BSA, and 50 μM of CPH-6 were used.

Fig. 9

Different CPHs showed different effects on OleT_SA activity (expressed as amount of styrene produced). Reaction condition: 5 μM OleT_SA, 2 mM hydrocinamic acid, 2 mM H₂O₂, 200 μM CPHs, room temperature for 30 min. Error bars represent standard deviations of duplicate experiments (n=2).