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.

Graphical abstract

Highlights

• •The interaction between cycloalkyl phosphorus heterocycles (CPHs) and P450 OleT_SA were studied by spectroscopic methods.
• •Dynamic mode of quenching mechanism was noticed in OleT_SA - CPHs interaction.
• •Hydrogen bond and van der Waals force were determined to contribute the conformation of OleT_SA- CPHs complex.
• •The penetration of CPH-6 into the interior environment of OleT_SA was proposed.
• •The effect of CPHs on OleT_SA enzyme activity was studied.

1. Introduction

Organophosphorus compounds widely exist in biological systems and have drawn largely attentions in medicinal application [[1], [2], [3], [4], [5], [6], [7], [8], [9]]. They have been used for the treatment of human diseases including Alzheimer's, schistosomiasis and osteoporosis [10,11]. Phosphorus heterocycles are heterocyclic compounds containing hetero (O, N) and phosphorus atoms. They have obtained widespread interests because of their potential pharmaceutic applications [[12], [13], [14], [15], [16]]. For example, the oxazaphosphorine ifosfamide (I, Fig. 1) is commonly used as an alkylating chemotherapeutic agent in oncology such as lung, cervical, testicular cancers, and bone or soft tissue sarcomas [12]. A group of new nonpeptidic cyclic phosphon-based hydroxamic acids (II, Fig. 1) have been employed as inhibitors of metalloproteinases [14]. In another example, benzoxaphosphol-2-ones (III, Fig. 1) were designed and prepared, which show significant antibacterial activities [15]. [1,2]oxaphosphinane (IV, Fig. 1) was reported to have an antiproliferative activity against a large panel of NCI cancer cell lines [16]. In addition, interactions of phosphorus heterocyclic compounds with biomacromolecules have been reported, suggesting important biological and pharmaceutical values of these derivatives [17,18].

Fig. 1.

Structure of representative phosphorus heterocycles as novel pharmaceuticals.

Cytochromes P450 (P450s) are widely found in a variety of species throughout the biosphere [19]. As a large family of heme-containing redox enzymes, they play an important role in the synthesis of many molecules such as steroid hormones, cholesterol and other fats and acids. Additional P450s serve to metabolize and detoxify exogenous compounds presented to the organism [[20], [21], [22], [23], [24]]. Studying the interaction between drugs and P450s is significant not only for drugs’ pharmacological characteristic analysis but also for the enzyme inhibitor/activator discovery [25]. Identification of effective binders to P450s is crucial in distinguishing the risk of drug-drug interactions, where a compound potentially influences the metabolism of other compounds [26]. Moreover, investigations on the interactions of small molecules and P450s will provide insights into the mechanisms of drug action as well as the design of novel medicines.

Fluorescence spectroscopy is a powerful tool to explore the interactions between small molecules and P450s, which can reveal valuable information of the binding features of small molecules to proteins. Several studies have been investigated, indicating that the fluorescence quenching of P450 enzymes could be triggered by a variety of molecules [[27], [28], [29], [30]]. Mitra and co-workers [27] employed steady-state and time-resolved fluorescence spectroscopy to study the binding of camphor in the active side of cytochrome P450cam. Shumyantseva et al. [28] utilized similar fluorescence assays to detect the existence of riboflavin binding site on cytochrome P450 2B4. In addition, cytochrome P450 CYP1A2 and CYP2D6 were selected to study their interaction with puerarin by Shao et al. by using different spectroscopic studies [29]. Guengerich and co-workers [30] focused on the binding relationship of 7,8-benzoflavone to human cytochrome P450 3A4, revealing the complex fluorescence quenching and binding mechanism.

Recently, we reported a novel method to synthesize a series of cycloalkyl phosphorus heterocycles (CPHs) [31]. Their biological applications are yet to be discovered. Interestingly, these molecules display specific fluorescence and environment responsive properties (e.g. polarity, as shown in Fig. S1), making them excellent probes for small molecule/P450s binding study. OleT is a new P450 enzyme first reported by Rude et al. [32] in 2011. Collective effort has been made to improve the enzyme's catalytic efficiency including the use of small non-polar molecules to stimulate its activity [[33], [34], [35]]. Detailed binding mechanisms are needed to further understand the stimulation effect. Herein, we studied the interactions between CPHs and P450 fatty acid decarboxylase OleT from Staphylococcus aureus (OleT_SA) by using fluorescence spectrum, UV–vis spectrum and ³¹P nuclear magnetic resonance (NMR) spectroscopies. The effect of CPHs on OleT_SA enzymatic activity was also tested. To the best of our knowledge, it is the first work to describe the interaction of phosphorous heterocycles with P450s, which will provide useful information for phosphorus heterocycles' potential biological application. The binding mechanisms illustrated in this work also guide us to design novel stimulator to leverage OleT.

2. Materials and methods

2.1. Materials

The CPHs used here were prepared according to the synthetic method reported previously by our group [31]. The substrate diaryl (arylethynyl)phosphine oxide 1 was reacted with commercially available cycloalkane 2 in the presence of di-tert-butyl peroxide (DTBP) (Fig. 2), delivering excellent yield of CPHs. The crude product was purified by chromatographic techniques. The stock solutions of synthesized molecules were prepared by dissolving them in dimethyl sulfoxide (DMSO) to make the concentration as 30 mM. A phosphate buffer (200 mM K₂HPO₄, 100 mM NaCl, pH 7.4) was used unless otherwise noted. Other chemicals used were of analytical reagent grade and double-distilled water was used throughout.

Fig. 2.

Synthesis of cycloalkyl phosphorus heterocycles (CPHs).

OleT_SA expression and purification were performed by following the published paper [36]. Briefly the kanamycin-resistant plasmid T5 containing the OleT_SA-His6 gene was transformed to BL21 (DE3) containing the chloramphenicol-resistant pTF2 plasmid which has GroEL/GroES/Tig chaperones encoded. A modified terrific broth (TB) (24 g/L yeast extract, 12 g/L tryptone, and 4 g/L peptone) supplemented with 50 μg/mL kanamycin and 20 μg/mL chloramphenicol was used for culturing. 125 mg/L thiamine, and trace metals, 100 μM isopropyl β-d-1-thiogalactopyranoside (IPTG), 10 ng/mL tetracycline and 10 mg/L 5-aminolevulinic acid were added for protein expression. The expression was made at 18 °C for 16–18 h. Nickel-nitriloacetic acid (Ni-NTA) affinity chromatography was used for the purification of the protein, followed by Butyl-S-Sepharose column [37]. Fractions with an Rz value (Abs418 nm/Abs280 nm) above 1.2 were pooled and dialyzed against 200 mM KPi buffer (pH 7.4). Proteins were stored at −80 °C until further use.

2.2. Apparatus

Fluorescence data were collected using Cary-Eclipse Fluorescence Spectrophotometer (Palo Alto, CA, USA) equipped with an attached Cary Temperature Controller. UV–vis spectra were carried on HP 8453 spectrophotometer (Palo Alto, CA, USA). The ³¹P NMR spectra were obtained with a Bruker Avance 400 spectrometer at 298 K (Bruker, Faellanden, Switzerland).

2.3. Methods

2.3.1. Fluorescence spectroscopy

Fluorescence spectra of OleT_SA (∼10 μM) were detected with varying the CPHs concentration (0, 5, 10, 15, 20, 25, 30 μM). With 280 nm excitation, the emission spectra were collected in the wavelength range from 300 to 500 nm at 293 K, 298 K, and 303 K, respectively. The different initial fluorescence intensity of the enzyme in all kinds of CPHs system were caused by the different concentration of OleT_SA. In addition, the fluorescence spectra of CPH-6 were carried out by keeping the concentration of CPH-6 at 30 μM, meanwhile varying the OleT_SA concentration (0, 0.2, 0.6, 2, 4, 6, 8 and 10 μM). The spectra were measured in the range of 270–500 nm at the excitation wavelength (λ_em) of 250 nm at 298 K. All the experiments were carried out in a conventional quartz cell using phosphate buffered saline (PBS).

2.3.2. UV–vis spectroscopy

Optical spectra were performed using 10 μM OleT_SA and the sequential addition of CPH-6 from the 30 mM stock solution. The absorption spectra were collected from 300 nm to 700 nm at 303 K.

2.3.3. ³¹P NMR study

CPH-6 (50 or 100 μM) in 600 μL D₂O was placed in a 10 mm NMR tube, followed by adding OleT_SA (50 μM) and the ³¹P NMR spectrum was recorded. Bovine serum albumin (BSA) was chosen as a protein control.

2.3.4. OleT_SA assay

The reaction mixtures (500 μL) containing 5 μM OleT_SA, 2 mM hydrocinnamic acid, 2 mM H₂O₂ in the presence and absence of 200 μM phosphorus heterocyclic compounds were incubated at room temperature for 30 min. The reactions were quenched by addition of 1% acetic acid followed by 1,2,3,4-tetramethylbenzene as the internal standard. After centrifuging at 12,000 r/min for 20 min, the supernatant was extracted and tested on HPLC.

3. Results and discussion

3.1. Fluorescence quenching analysis

Intrinsic fluorescence of proteins is caused by three amino acids residues (tryptophan, tyrosine, and phenylalanine) containing aromatic groups. Among them, the tryptophan residues are believed to be main contributors [38]. A very significant aspect of proteins' intrinsic fluorescence is that its tryptophan residues have strong sensitivity to local environment [[39], [40], [41]]. The changes of fluorescence of tryptophan have been a useful tool to reveal the process of protein folding, aggregation and conformation transition [40]. As a result, the proteins' intrinsic fluorescence spectrum responding to small molecules can reflect valuable information about their interactions. In our study, the analysis of OleT_SA fluorescence with increasing concentration of all derivatives (0, 5, 10, 15, 20, 25, and 30 μM, respectively) at 298 K was monitored. As shown in Fig. 3, decrease of fluorescence intensity was observed for all derivatives, indicating that the interaction between OleT_SA and the CPHs can quench the enzyme's fluorescence signal. In addition, many CPHs in our research showed fluorescence signal at 400–500 nm with 280 nm excitation. In Fig. 3, with higher concentration of CPH-1, a stronger fluorescence signal was observed at 450 nm.

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.

To gain insights into the interaction mechanism of CPHs with OleT_SA, CPH-6 was chosen as a model for further investigation. The emission spectra of the compound CPH-6 with various concentrations of OleT_SA at 298 K were collected (Fig. 4A). Under 250 nm excitation, CPH-6 emitted fluorescence signal in the range of 380–440 nm, which decreased with the increasing concentration of OleT_SA. The result indicated that the fluorescence of the molecule could also be quenched by OleT_SA, which could be attributed to the highly hydrophobic environment of the enzyme pocket.

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.

The fluorescence data were further analyzed to reveal the origins of the quenching. It is known that dynamic quenching is the process of inactivation of the excited state of fluorophores after interaction with quenching molecules [42]. However, static quenching often generates nonfluorescent complex of the fluorophore and the quencher. The discrimination of dynamic and static quenching depends on the relationship of quenching constant and temperature [42]. The dynamic quenching constant increases with the rise of temperature while the other one shows the decline of quenching constant with the increase of temperature. The Stern-Volmer equation (Eq. (1)) can be applied to explain the quenching mechanism.

$$\frac{F_0}{F}=1+K_q{\tau }_0\left[OleT\right]=1+K_{sv}[OleT]$$ (1)

Here, F₀ and F represent the fluorescence intensities of OleT without and with a quencher, [OleT] refers to the concentration of OleT, K_q means the biomolecular quenching rate constant, K_SV is the Stern-Volmer constant, τ₀ is the average lifetime of the molecule without quencher. The average fluorescence lifetime of the biomolecule is evaluated at about 10 ns [43,44], so the quenching constant K_q could be obtained according to the Eq. (2).

$$K_q{\tau }_0=K_{sv}$$ (2)

The plots of F₀/F versus [OleT] are shown in Fig. 4B. The values of K_SV and K_q were determined by the slope of the plots and Eq. (2). The results are listed in Table 1 together with the correlation coefficients. As shown in Table 1, a significant increase in Stern-Volmer constant values was observed with increasing temperatures, indicating that the dynamic quenching was responsible for the quenching process of OleT_SA and CPH-6.

Table 1.

Stern-Volmer quenching constants of OleT_SA and CPH-6 at 293 K, 298 K, and 303 K.

System	Temp. (K)	Ksv (M−1) × 10−2	Kq (M−1s−1) × 106	Ra
OleTSA-CPH-6	293	11.1	11.1	0.9786
OleTSA-CPH-6	298	16.9	16.9	0.9777
OleTSA-CPH-6	303	20.1	20.1	0.9523

^aCorrelation coefficient.

The binding constant (K) for OleT interaction with CPH-6 and the number of binding sites were described by double logarithmic plots as follows (Eq. (3)) [45,46].

$$\text{log}\frac{F_0-F}{F}=\log K+n\text{log}\left[OleT\right]$$ (3)

Here, F₀ and F represent the fluorescence intensities of OleT without and with a quencher, K denotes the binding constant of OleT_SA with CPH-6, and n represents the number of binding sites. The characteristic binding parameters K and n were obtained from the plots of log ((F₀–F)/F) versus log [OleT] (Fig. 5).

Fig. 5.

Double logarithmic plots for OleT_SA-CPH-6 complexes.

As shown in Table 2, the value of n at the tested temperatures was around equal to one, which demonstrated that there was a single set equivalent binding site in OleT_SA with CPH-6. In addition, the value of K is estimated to be 15.2 × 10⁴ M⁻¹ at 293 K, suggesting a moderately binding affinity between these two substances. A decrease was obtained in the value of K with the increase of temperature from 293 K to 303 K, indicating that the protein can better accommodate the molecule at 293 K than the other two temperatures and the stability between them decreases with increased temperature.

Table 2.

Binding constants and the number of binding sites on OleT_SA for CPH-6.

System	Temp. (K)	K (M−1) × 104	n	Ra
OleTSA-CPH-6	293	15.2	1.25	0.9805
OleTSA-CPH-6	298	0.40	0.86	0.9788
OleTSA-CPH-6	303	0.28	0.79	0.9693

^aCorrelation coefficient.

3.2. Thermodynamic analysis

It is well studied that non-covalent interactive forces including hydrogen bonding, hydrophobic interactions, van der Waals force and electrostatic attraction contribute to the binding of small molecules with biomacromolecules [[47], [48], [49]]. Ross and Subramanian [47] reported the main binding forces based on the sign and magnitude of thermodynamic parameters such as enthalpy change (ΔH) and entropy change (ΔS). The principle includes: a) ΔH > 0 and ΔS > 0, hydrophobic forces; b) ΔH < 0 and ΔS < 0, van der Waals interactions and hydrogen bonds; c) ΔH < 0 and ΔS > 0, electrostatic interactions. The ΔH and ΔS are calculated from the liner Van't Hoff plot (Eq. (4)).

$$lnK=-\frac{\mathrm{\Delta }H}{RT}+\frac{\mathrm{\Delta }S}{R}$$ (4)

Here, K shows the associative binding constant at the specific temperature and R is the gas constant. The tested temperatures are 293, 298, and 303 K. ΔH and ΔS are obtained from the slope and intercept of the plot of lnK versus 1/T (Fig. 6). Free energy change (ΔG) is estimated from the following relationship (Eq. (5)).

$$\mathrm{\Delta }G=\mathrm{\Delta }H-T\mathrm{\Delta }S=-RTlnK$$ (5)

Fig. 6.

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

As shown in Table 3, all the thermodynamic parameters ΔG, ΔH and ΔS were negative at the tested temperatures. Negative values of free energy (ΔG) revealed that the binding of OleT_SA with CPH-6 was spontaneous. Both the values of ΔH and ΔS were negative, indicating that the main forces in the OleT_SA-CPH-6 complex binding were hydrogen bond and van der Waals force.

Table 3.

Thermodynamic parameters for the interaction of OleT_SA with CPH-6 at tested temperature.

System	Temp. (K)	ΔH (kJ/mol)	ΔS (kJ/mol∙K)	ΔG (kJ/mol)
OleTSA-CPH-6	293	−296.1	−915.7	−29.1
OleTSA-CPH-6	298	−296.1	−915.7	−20.6
OleTSA-CPH-6	303	−296.1	−915.7	−20.0

3.3. UV–vis spectrum analysis

It has been well established that UV–vis spectra of P450s are sensitive to the redox state of the heme as well as its local environment [50]. To further study the binding effect of CPH-6 to OleT_SA, the spectrum of OleT_SA was monitored by UV–vis with or without of CPH-6. As shown in Fig. 7, the fluorescence intensity of OleT_SA with 25 μM or 50 μM CPH-6 was adjusted by the extraction of the background fluorescence of corresponding concentration of substrate to eliminate the interference. It was found that the substrate-free OleT_SA was in the mixed state at the beginning and the addition of 25 μM CPH-6 led to a slight increase of high spin. With a higher concentration (50 μM), this increment went further, indicating that CPH-6 can alter the microenvironment of heme iron of OleT_SA.

Fig. 7.

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

3.4. ³¹P NMR study

The interactions described above between CPH-6 and OleT_SA were confirmed by ³¹P NMR analysis. As shown in Fig. 8, for the OleT_SA-CPH-6 complex with 1:1 M ratio, ³¹P chemical shift value was moved to high field compared to the pure molecule CPH-6, suggesting that the environment of CPH-6 changed after adding into OleT_SA. As a control, the ³¹P chemical shift of CPH-6 did not change after incubation with BSA in the same condition. With increased OleT_SA concentration (i.e., OleT_SA-CPH-6 complex with 2:1 M ratio), further shift was observed (Fig. S2). These results confirmed the interaction between OleT_SA and CPH-6.

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.

3.5. OleT_SA enzymatic activity assay

OleT_SA catalyzes the decarboxylation of hydrocinnamic acid to form styrene [51], which can be easily detected by LC. The effect of phosphorous heterocycles on OleT_SA activity was characterized by using hydrocinnamic acid as substrate (Fig. 9). Interestingly, the CPHs with different side groups showed different effects on OleT_SA activity. For example, OleT_SA showed around 30% enhanced activity with treatment by CPH-5 (cyclohexyl phosphorous heterocycle with fluoro group) while around 20% activity was inhibited by CPH-3 (cyclopentyl phosphorous heterocycle). Further characterization of enzyme-CPHs complex structure will provide useful information to understand these effects. Nevertheless, the different effects resulting from the fine-tuned small molecule structure represent a viable way to leverage enzyme activity. Such information can be also used for P450 enzyme effector design.

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).

4. Conclusions

In conclusion, this research was to study the interactions between CPHs and OleT_SA by fluorescence spectra, UV–vis and ³¹P NMR spectroscopies. Dynamic quenching mechanism was identified. The binding constant value of 15.2 × 10⁴ M⁻¹ at 293 K indicated a moderately binding affinity between CPHs and OleT_SA. Hydrogen bonding and van der Waals force were identified as main forces involved in the interactions on the basis of thermodynamic parameters analysis. UV–vis spectroscopies suggested that the presence of CPHs could affect the enzyme's interior environment. The ³¹P NMR studies revealed that binding of CPHs to OleT_SA had an influence on the chemical shift of ³¹P NMR of the molecule. Moreover, the effect of such molecules on OleT_SA enzymatic activity indicated potential functions of these molecules and provided useful information for enzyme stimulator design.

Declaration of competing interest

The authors declare that there have no conflicts of interest.

Appendix A. Supplementary data

The following is the Supplementary data to this article: