Structure and biochemical properties of the alkene producing cytochrome P450 OleTJE (CYP152L1) from the Jeotgalicoccus sp. 8456 bacterium

Abstract

The production of hydrocarbons in nature has been documented for only a limited set of organisms, with many of the molecular components underpinning these processes only recently identified. There is an obvious scope for application of these catalysts and engineered variants thereof in the future production of biofuels. Here we present biochemical characterization and crystal structures of a cytochrome P450 fatty acid peroxygenase: the terminal alkene forming OleTJE (CYP152L1) from Jeotgalicoccus sp. 8456. OleTJE is stabilized at high ionic strength, but aggregation and precipitation of OleTJE in low salt buffer can be turned to advantage for purification, because resolubilized OleTJE is fully active and extensively dissociated from lipids. OleTJE binds avidly to a range of long chain fatty acids, and structures of both ligand-free and arachidic acid-bound OleTJE reveal that the P450 active site is preformed for fatty acid binding. OleTJE heme iron has an unusually positive redox potential (-103 mV versus normal hydrogen electrode), which is not significantly affected by substrate binding, despite extensive conversion of the heme iron to a high spin ferric state. Terminal alkenes are produced from a range of saturated fatty acids (C12-C20), and stopped-flow spectroscopy indicates a rapid reaction between peroxide and fatty acid-bound OleTJE (167 s(-1) at 200 μm H2O2). Surprisingly, the active site is highly similar in structure to the related P450BSβ, which catalyzes hydroxylation of fatty acids as opposed to decarboxylation. Our data provide new insights into structural and mechanistic properties of a robust P450 with potential industrial applications.

Keywords: Biofuel; Crystal Structure; Cytochrome P450; Decarboxylase; Electron Paramagnetic Resonance (EPR); Enzyme Kinetics; Enzyme Mechanisms; Fatty Acid-binding Protein; Hydrogen Peroxide; X-ray Crystallography.

FIGURE 1.

The cytochrome P450 catalytic cycle. The reaction sequence starts at the top, with the P450 “resting” state having a ferric (Fe³⁺) 6-coordinate low spin heme iron axially coordinated by cysteine thiolate (S) and a weakly bound water molecule (H₂O). Binding of substrate (RH) displaces the water ligand to leave a 5-coordinate high spin heme iron. This is reduced by a redox partner, and the ferrous iron then binds dioxygen to form a ferric-superoxo complex. A further single electron reduction by the redox partner generates the ferric-peroxo intermediate, which is protonated to form the transient ferric-hydroperoxo (compound 0) species. Compound 0 is further protonated and dehydrated to form the ferryl-oxo porphyrin radical cation species compound I. The compound I is considered to be the major oxidant in P450 reactions and abstracts a hydrogen from RH to produce a substrate radical, prior to “rebounding” the hydroxyl to the substrate to form hydroxylated product (ROH) and to restore the resting state of the P450 (8, 55). The double-headed arrow crossing the cycle between the substrate-bound ferric P450 and compound 0 describes the catalytic mechanism for OleT_JE and related peroxygenase P450s (e.g. P450_BSβ and P450_SPα) (16–18). Direct interaction of OleT_JE with H₂O₂ produces a reactive iron-oxo species (compound 0) that is further protonated and dehydrated to form compound I, leading to either fatty acid substrate decarboxylation (major route) or fatty acid hydroxylation (minor route), as described under “Discussion” section and in the legend to Fig. 13.

FIGURE 2.

Phylogenetic tree for OleT_JE and other members of the CYP152 P450 family. The tree shows the relationship between members of the bacterial CYP152 family and between the CYP152s and other bacterial cytochrome P450 families. CYP152L1 (OleT_JE) is most closely related to CYP152L2 from S. massiliensis S46 (64% amino acid identity).

FIGURE 3.

Purification of OleT_JE. Proteins are resolved on a 12% SDS-polyacrylamide gel. The first lane shows markers of the indicated sizes (Fermentas PageRuler Plus prestained protein marker, Thermo Scientific). Purified non-tagged OleT_JE (∼1.5 μg) is shown in lane 2 and shows protein isolated from E. coli following steps of Ni-IDA chromatography, protein precipitation and resolubilization, His tag removal by proteolysis using HRV 3C, and passage through a nickel-Sepharose column.

FIGURE 4.

UV-visible spectroscopic features of OleT_JE. UV-visible absorption spectra of OleT (4.9 μm) are shown for (i) the oxidized (ferric), substrate-free form (solid back line) with Soret maximum at 418 nm, (ii) the sodium dithionite-reduced (ferrous) form (dashed line) with Soret maximum at 414 nm, (iii) the ferrous-CO complex (dotted line) with the Soret band shifted to 449 nm (the P450 form), and (iv) the ferric-NO complex (dashed and dotted line) with Soret maximum at ∼427 nm. Inset, magnification of the Q-band region for the same OleT_JE spectra, highlighting changes observed on OleT_JE reduction and on formation of its CO and NO complexes. Lines are identified in the same way as in the main panel.

FIGURE 5.

Analysis of fatty acid and dithiothreitol binding to OleT_JE. A, a spectral titration for OleT_JE (9.8 μm) with arachidic acid (C20:0). Arrows indicate the progressive decrease in the ferric LS Soret band (at 418 nm) and the concomitant increase in the ferric HS feature at 394 nm. The development of a small thiolate-to-HS ferric charge transfer band is seen at ∼650 nm as the titration progresses. The inset shows a fit (using the Morrison equation) of arachidic acid-induced Soret absorbance change versus fatty acid concentration, yielding a K_d value of 0.29 ± 0.05 μm for arachidic acid. B, spectra for an OleT_JE (9.8 μm) titration with lauric acid (C12:0). In this case, HS heme development is less extensive than for arachidic acid, and the K_d value (inset) is 0.77 ± 0.02 μm. C, titration of OleT_JE (6.1 μm) with DTT. The binding of DTT is associated with the splitting of the heme signal into two distinct features: a hyperporphyrin (split Soret) spectrum with maxima at 372 and ∼460 nm and a distinct Soret feature at 423 nm. The former results from distal coordination of the OleT_JE by DTT thiolate (trans to cysteine thiolate), whereas the latter has DTT thiol as the distal ligand (23). The inset shows a plot of DTT-induced Soret absorbance shift versus DTT concentration, fitted using a hyperbolic equation to yield a K_d value of 159 ± 7 μm. Data were collected, processed, and fitted as described under “Experimental Procedures.”

FIGURE 6.

Determination of the OleT_JE heme iron reduction potential in its substrate-free and arachidic acid-bound forms. A, data from a spectroelectrochemical redox titration of ligand-free OleT_JE (8.1 μm). The spectrum for the oxidized enzyme (solid line) shows the Soret maximum at 419 nm, whereas that for the fully dithionite-reduced P450 (dashed line) has its Soret maximum at 406 nm and shows a single feature in the Q-band region at ∼560 nm. Intermediate spectra are shown in dotted lines. Arrows, direction of absorption changes observed during the reductive part of the titration. Inset, plot of absorbance at the Soret peak (417 nm) versus the applied potential corrected for the NHE. Data are fitted using the Nernst equation to give a midpoint potential of E⁰′ = −103 ± 6 mV. B, redox titration for arachidic acid-bound OleT_JE (8.1 μm; thick solid line). The oxidized substrate-bound species has its Soret maximum at 395 nm, and the fully reduced form (thick dotted line) has a maximum at ∼420 nm. Intermediate spectra in the titration are shown in dotted lines. Arrows again indicate absorption changes observed during the reductive part of the titration. The inset shows a plot of absorbance at the substrate-bound Soret peak (395 nm) versus the applied potential corrected for the NHE, with data fitted using the Nernst equation to yield E⁰′ = −105 ± 6 mV.

FIGURE 7.

Stopped-flow kinetics of H₂O₂-dependent oxidation of substrate-bound OleT_JE. A, plot of the observed rate constants (k_obs) for H₂O₂ binding to OleT_JE (and concomitant substrate oxidation) versus the H₂O₂ concentration. Data were measured at 417 nm, reflecting recovery of the LS OleT_JE form. The k_obs versus [H₂O₂] data were fitted to a linear function to yield the second order rate constant of H₂O₂ binding/substrate (arachidic acid) oxidation of k_on = 0.80 ± 0.02 μm⁻¹ s⁻¹, k_off = 8.32 ± 1.96 s⁻¹, and an apparent K_d value of 10.40 ± 2.71 μm for H₂O₂ binding, derived from k_off/k_on. B, stopped-flow PDA data observed at 7.58 μm H₂O₂, demonstrating the OleT_JE spectral conversion from the HS, substrate-bound form (Soret maximum at 394 nm) to the LS reoxidized state (Soret maximum at ∼417 nm) upon mixing with H₂O₂. The inset shows the corresponding plot of the absorbance data at 417 nm (open circles) against time. The data were fitted using a single exponential function to yield an apparent rate constant of 12.50 ± 1.16 s⁻¹.

FIGURE 8.

EPR spectroscopic analysis of OleT_JE. X-band continuous wave EPR spectra (see “Experimental Procedures” for acquisition parameters) are shown for purified OleT_JE (A) and OleT_JE plus arachidic acid (B), with both enzymes purified using Method 1 (see “Experimental Procedures”). The g values are marked in each case. The OleT_JE concentration is 305 μm for substrate-free enzyme and 205 μm for arachidic acid-bound OleT_JE, with arachidic acid added to a saturating concentration.

FIGURE 9.

Oxidative decarboxylation of arachidic acid and lauric acid by OleT_JE. A, total ion count from GC separation of the C19 terminal alkene 1-nonadecene in the reaction of OleT_JE with arachidic acid (C20:0) (top), with mass spectrometric analysis of the major peak at 16.21 min confirming its identity (bottom; inset highlighting the region of the 1-nonadecene mass ion with m/z = 266). B, total ion count from GC separation of the C11 terminal alkene 1-undecene following reaction of OleT_JE with lauric acid (C12:0) (top), with mass spectrometric analysis of the major peak at 6.63 min confirming its identity (bottom; inset highlighting the region of the 1-undecene mass ion with m/z = 154).

FIGURE 10.

Comparison between the OleT_JE and P450_BSβ P450 structures. An overlay is shown for the substrate-bound forms of OleT_JE (in blue; PDB code 4L40) and P450_BSβ (in gray; PDB code 1IZO) in a schematic representation. The bound substrates and heme groups are represented in sticks, colored in magenta for the OleT_JE arachidic acid-bound form and in green for the palmitic acid-bound form of P450_BSβ.

FIGURE 11.

Comparison of the OleT_JE and P450_BSβ fatty acid binding modes. The image shows a side by side overlay of the OleT_JE and P450_BSβ fatty acid-bound active sites, with key residues contacting the substrates shown. For clarity, main chain atoms have been removed. The left panel depicts OleT_JE in atom colored sticks, with the corresponding P450_BSβ residues shown in gray lines. The right panel shows the same view but with the P450_BSβ residues in atom colored sticks and OleT_JE shown in gray lines. Substrates are arachidic acid (C20:0) for OleT_JE and palmitic acid (C16:0) for P450_BSβ.

FIGURE 12.

Detailed view of the OleT_JE active site. Left, active site region for the ligand-free OleT_JE structure (key amino acids shown in atom colored sticks with green carbons), with water molecules close to the iron shown as red spheres. Right, the same region in the same orientation for the ligand-bound OleT_JE structure (key amino acids in blue), with the C20 ligand in magenta sticks. The omit map for the ligand is shown as a green mesh contoured at 3 σ.

FIGURE 13.

Proposed mechanism for OleT_JE. The cytochrome P450 OleT_JE catalyzes oxidative decarboxylation of long chain fatty acids as its major reaction, whereas the highly related P450_BSβ produces predominantly β-hydroxylated fatty acids from the same substrates (16, 17). OleT_JE His85 (replaced by Gln-85 in P450_BSβ) is proposed to act as a proton donor to the ferryl-oxo porphyrin radical cation (compound I) intermediate in this P450, concomitant with its reduction to compound II by an electron abstracted from the fatty acid carboxylate. Homolytic scission of the C–C_α bond, concomitant with hydrogen abstraction from C_α to compound II, leads to production of the terminal alkene and CO₂. For the hydroxylase P450_BSβ, this reaction cannot occur in the absence of an appropriate proton donor to compound II, with the catalytic outcome instead being a typical P450 hydroxylation at the C_β position.