Oxidation of 10-undecenoic acid by cytochrome P450(BM-3) and its Compound I transient

Oxidations of 10-undecenoic acid by cytochrome P450(BM-3) and its Compound I transient were studied. The only product formed in Compound I oxidations was 10,11-epoxyundecanoic acid, whereas the enzyme under turnover conditions gave the epoxide and 9-hydroxy-10-undecenoic acid in a 10 : 90 ratio. Kinetic studies at 0 °C of oxidations by Compounds I formed by MCPBA oxidation and by a photo-oxidation pathway gave the same results, displaying saturation kinetics that yielded equilibrium binding constants and first-order oxidation rate constants that were experimentally indistinguishable. Oxidation of 10-undecenoic acid by Compound I from CYP119 generated by MCBPA oxidation also gave 10,11-epoxyundecanoic acid as the only product. CYP119 Compound I bound the substrate less strongly but reacted with a faster oxidation rate constant than P450(BM-3) Compound I. The kinetic parameters for oxidation of the substrate by P450(BM-3) under turnover conditions were similar to those of the Compound I transient even though the products differed.     

Directed evolution of the fatty-acid hydroxylase P450 BM-3 into an indole-hydroxylating catalyst

The self-sufficient cytochrome P450 BM-3 enzyme from Bacillus megaterium catalyzes subterminal hydroxylation of saturated long-chain fatty acids and structurally related compounds. Since the primary structure of P450 BM-3 is homologous to that of mammalian P450 type II, it represents an excellent model for this family of enzymes. During studies on the directed evolution of P450 BM-3 into a medium-chain fatty-acid hydroxylase, several mutants, in particular the triple mutant Phe87Val, Leu188Gln, Ala74Gly, were observed to hydroxylate indole, producing indigo and indirubin at a catalytic efficiency of 1365 M(-1)s(-1) (kcat=2.73 s(-1) and Km=2.0 mM). Both products were unequivocally characterized by NMR and MS analysis. Wild-type P450 BM-3 is incapable to hydroxylate indole. These results demonstrate that an enzyme can be engineered to catalyze the transformation of substrates with structures widely divergent from those of its native substrate.     

Enantioselective alpha-hydroxylation of 2-arylacetic acid derivatives and buspirone catalyzed by engineered cytochrome P450 BM-3

Here we report that an engineered microbial cytochrome P450 BM-3 (CYP102A subfamily) efficiently catalyzes the alpha-hydroxylation of phenylacetic acid esters. This P450 BM-3 variant also produces the authentic human metabolite of buspirone, R-6-hydroxybuspirone, with 99.5% ee.     

Resonance surface plasmon spectroscopy: low molecular weight substrate binding to cytochrome p450

A new detection mechanism has been developed for low molecular weight substrate binding to heme proteins based on resonance localized surface plasmon spectroscopy. Cytochrome P450 has strong electronic transitions in the visible wavelength region. Upon binding of a substrate molecule (e.g., camphor), the absorption band of cytochrome P450 shifts to shorter wavelength. The event of camphor binding to a nanoparticle surface modified with cytochrome P450 protein receptors is monitored using UV-vis spectroscopy. It is observed for the first time that the binding of the substrate molecules to the protein receptor induces a blue-shift in the localized surface plasmon resonance (LSPR) of the nanosensors. The coupling between the molecular resonance of the substrate-free and substrate-bound cytochrome P450 proteins and the nanoparticles' LSPR leads to a highly wavelength-dependent LSPR response. When the LSPR of the nanoparticles is located at a wavelength distant from the cytochrome P450 resonance, an average of approximately 19 nm red-shift is observed upon cytochrome P450 binding to the nanoparticles and a approximately 6 nm blue-shift is observed upon camphor binding However, this response is significantly amplified approximately 3 to 5 times when the LSPR of the nanoparticles is located at a slightly longer wavelength than the cytochrome P450 resonance, that is, a 66.2 nm red-shift upon cytochrome P450 binding and a 34.7 nm blue-shift upon camphor binding. This is the first example of the detection of small molecules binding to a protein modified nanoparticle surface on the basis of LSPR.     

Stereoselective hydroxylation of an achiral cyclopentanecarboxylic acid derivative using engineered P450s BM-3

Substrate engineered, achiral carboxylic acid derivative was biohydroxylated with various mutants of cytochrome P450 BM-3 to give two out of the four possible diastereoisomers in high de and ee. The BM-3 mutants exhibit up to 9200 total turnovers for hydroxylation of the engineered substrate, which without the protecting group is not transformed by this enzyme.     

Molecular modeling of cytochrome P450 3A4

The three-dimensional structure of human cytochrome P450 3A4 was modeled based on crystallographic coordinates of four bacterial P450s: P450 BM-3, P450cam, P450terp, and P450eryF. The P450 3A4 sequence was aligned to those of the known proteins using a structure-based alignment of P450 BM-3, P450cam, P450terp, and P450eryF. The coordinates of the model were then calculated using a consensus strategy, and the final structure was optimized in the presence of water. The P450 3A4 model resembles P450 BM-3 the most, but the B helix is similar to that of P450eryF, which leads to an enlarged active site when compared with P450 BM-3, P450cam, and P450terp. The 3A4 residues equivalent to known substrate contact residues of the bacterial proteins and key residues of rat P450 2B1 are located in the active site or the substrate access channel. Docking of progesterone into the P450 3A4 model demonstrated that the substrate bound in a 6-orientation can interact with a number of active site residues, such as 114, 119, 301, 304, 305, 309, 370, 373, and 479, through hydrophobic interactions. The active site of the enzyme can also accommodate erythromycin, which, in addition to the residues listed for progesterone, also contacts residues 101, 104, 105, 214, 215, 217, 218, 374, and 478. The majority of 3A4 residues which interact with progesterone and/or erythromycin possess their equivalents in key residues of P450 2B enzymes, except for residues 297, 480 and 482, which do not contact either substrate in P450 3A4. The results from docking of progesterone and erythromycin into the enzyme model make it possible to pinpoint residues which may be important for 3A4 function and to target them for site-directed mutagenesis.     

Biotransformation of the sesquiterpene (+)-valencene by cytochrome P450cam and P450BM-3

The sesquiterpenoids are a large class of naturally occurring compounds with biological functions and desirable properties. Oxidation of the sesquiterpene (+)-valencene by wild type and mutants of P450cam from Pseudomonas putida, and of P450BM-3 from Bacillus megaterium, have been investigated as a potential route to (+)-nootkatone, a fine fragrance. Wild type P450cam did not oxidise (+)-valencene but the mutants showed activities up to 9.8 nmol (nmol P450)(-1) min(-1), with (+)-trans-nootkatol and (+)-nootkatone constituting >85% of the products. Wild type P450BM-3 and mutants had higher activities (up to 43 min(-1)) than P450cam but were much less selective. Of the many products, cis- and trans-(+)-nootkatol, (+)-nootkatone, cis-(+)-valencene-1,10-epoxide, trans-(+)-nootkaton-9-ol, and (+)-nootkatone-13S,14-epoxide were isolated from whole-cell reactions and characterised. The selectivity patterns suggest that (+)-valencene has one binding orientation in P450cam but multiple orientations in P450BM-3.     

P450 monooxygenase in biotechnology. I. Single-step, large-scale purification method for cytochrome P450 BM-3 by anion-exchange chromatography.

An efficient single-step purification protocol for recombinant cytochrome P450 BM-3 from Bacillus megaterium, expressed in E. coli, was developed. Functional crude protein was obtained by disintegrating induced E. coli DH5 alpha and removing cell debris by centrifugation. After investigating different anion-exchange matrices, elution salts and the elution procedures involving an AKTAexplorer system, adsorption of the crude extract from lysed E. coli to Toyopearl DEAE 650M anion exchanger, followed by a two-step elution using NaCl, proved sufficient to isolate almost pure protein without inactivation (up to 93% P450 BM-3 content) in yields that ranged between 79-86%. The purification method could be scaled up 1500-fold and higher without further optimization to a 6-1 production-scale column containing Toyopearl DEAE 650M anion exchanger.     

Metal-substituted protein MRI contrast agents engineered for enhanced relaxivity and ligand sensitivity

Engineered metalloproteins constitute a flexible new class of analyte-sensitive molecular imaging agents detectable by magnetic resonance imaging (MRI), but their contrast effects are generally weaker than synthetic agents. To augment the proton relaxivity of agents derived from the heme domain of cytochrome P450 BM3 (BM3h), we formed manganese(III)-containing proteins that have higher electron spin than their native ferric iron counterparts. Metal substitution was achieved by coexpressing BM3h variants with the bacterial heme transporter ChuA in Escherichia coli and supplementing the growth medium with Mn3+-protoporphyrin IX. Manganic BM3h variants exhibited up to 2.6-fold higher T1 relaxivities relative to native BM3h at 4.7 T. Application of ChuA-mediated porphyrin substitution to a collection of thermostable chimeric P450 domains resulted in a stable, high-relaxivity BM3h derivative displaying a 63% relaxivity change upon binding of arachidonic acid, a natural ligand for the P450 enzyme and an important component of biological signaling pathways. This work demonstrates that protein-based MRI sensors with robust ligand sensitivity may be created with ease by including metal substitution among the toolkit of methods available to the protein engineer.     

Alkene epoxidation catalyzed by cytochrome P450 BM-3 139-3

We recently reported conversion of cytochrome P450 BM-3, a medium-chain (C₁₂-C₁₈) fatty acid monooxygenase, into a highly efficient alkane hydroxylase by directed evolution [Nat. Biotechnol. 2002, 20, 1135]. P450 BM-3 mutant 139-3 exhibited high activity towards a variety of fatty acid and alkane substrates, including C₃-C₈ alkanes. We report here that mutant 139-3 is also active on benzene, styrene, cyclohexene, 1-hexene, and propylene. Benzene is converted to phenol, while styrene is converted to styrene oxide. Propylene oxidation generates only propylene oxide, but cyclohexene oxidation produces a mixture of cyclohexene oxide (85%) and 2-cyclohexene-1-ol (15%), and 1-hexene is converted to the allylic hydroxylation product, 1-hexene-3-ol. Initial rates of NADPH oxidation for 139-3 in the presence of the substrates greatly (17- to >100-fold) surpass the wild-type in all cases. However, NADPH consumption is only partially coupled to product formation (14-79%). This cytochrome P450 epoxidation catalyst is a suitable starting point for further evolution to improve coupling and activity.     

Cytochrome‐P450‐Induced Ordering of Microsomal Membranes Modulates Affinity for Drugs

Although membrane environment is known to boost drug metabolism by mammalian cytochrome P450s, the factors that stabilize the structural folding and enhance protein function are unclear. In this study, we use peptide‐based lipid nanodiscs to “trap” the lipid boundaries of microsomal cytochrome P450 2B4. We report the first evidence that CYP2B4 is able to induce the formation of raft domains in a biomimetic compound of the endoplasmic reticulum. NMR experiments were used to identify and quantitatively determine the lipids present in nanodiscs. A combination of biophysical experiments and molecular dynamics simulations revealed a sphingomyelin binding region in CYP2B4. The protein‐induced lipid raft formation increased the thermal stability of P450 and dramatically altered ligand binding kinetics of the hydrophilic ligand BHT. These results unveil membrane/protein dynamics that contribute to the delicate mechanism of redox catalysis in lipid membrane.     

Substrate binding favors enhanced NO binding to P450cam

Ferric cytochrome P450cam from Pseudomonas putida (P450cam) in buffer solution at physiological pH 7.4 reversibly binds NO to yield the nitrosyl complex P450cam(NO). The presence of 1R-camphor affects the dynamics of NO binding to P450cam and enhances the association and dissociation rate constants significantly. In the case of the substrate-free form of P450cam, subconformers are evident and the NO binding kinetics are much slower than in the presence of the substrate. The association and dissociation processes were investigated by both laser flash photolysis and stopped-flow techniques at ambient and high pressure. Large and positive values of S and V observed for NO binding to and release from the substrate-free P450cam complex are consistent with the operation of a limiting dissociative ligand substitution mechanism, where the lability of coordinated water dominates the reactivity of the iron(III)-heme center with NO. In contrast, NO binding to P450cam in the presence of camphor displays negative activation entropy and activation volume values that support a mechanism dominated by a bond formation process. Volume profiles for the binding of NO appear to be a valuable approach to explain the differences observed for P450cam in the absence and presence of the substrate and enable the clarification of the underlying reaction mechanisms at a molecular level. Changes in spin state of the iron center during the binding/release of NO contribute significantly to the observed volume effects. The results are discussed in terms of relevance for the biological function of cytochrome P450 and in context to other investigations of the related reactions between NO and imidazole- and thiolate-ligated iron(III) hemoproteins.     

Enantioselective epoxidation of terminal alkenes to (R)- and (S)-epoxides by engineered cytochromes P450 BM-3

Cytochrome P450 BM-3 from Bacillus megaterium was engineered for enantioselective epoxidation of simple terminal alkenes. Screening saturation mutagenesis libraries, in which mutations were introduced in the active site of an engineered P450, followed by recombination of beneficial mutations generated two P450 BM-3 variants that convert a range of terminal alkenes to either (R)- or (S)-epoxide (up to 83 % ee) with high catalytic turnovers (up to 1370) and high epoxidation selectivities (up to 95 %). A biocatalytic system using E. coli lysates containing P450 variants as the epoxidation catalysts and in vitro NADPH regeneration by the alcohol dehydrogenase from Thermoanaerobium brockii generates each of the epoxide enantiomers, without additional cofactor.     

Ligand-induced conformational heterogeneity of cytochrome P450 CYP119 identified by 2D NMR spectroscopy with the unnatural amino acid (13)C-p-methoxyphenylalanine

Conformational dynamics are thought to play an important role in ligand binding and catalysis by cytochrome P450 enzymes, but few techniques exist to examine them in molecular detail. Using a unique isotopic labeling strategy, we have site specifically inserted a (13)C-labeled unnatural amino acid residue, (13)C-p-methoxyphenylalanine (MeOF), into two different locations in the substrate binding region of the thermophilic cytochrome P450 enzyme CYP119. Surprisingly, in both cases the resonance signal from the ligand-free protein is represented by a doublet in the (1)H,(13)C-HSQC spectrum. Upon binding of 4-phenylimidazole, the signals from the initial resonances are reduced in favor of a single new resonance, in the case of the F162MeOF mutant, or two new resonances, in the case of the F153MeOF mutant. This represents the first direct physical evidence for the ligand-dependent existence of multiple P450 conformers simultaneously in solution. This general approach may be used to further illuminate the role that conformational dynamics plays in the complex enzymatic phenomena exhibited by P450 enzymes.     

Preparation, characterization, and substrate metabolism of gold-immobilized cytochrome P450 2C9

The cytochrome P450 enzymes represent an important class of heme-containing enzymes. There is considerable interest in immobilizing these enzymes on a surface so that interactions between a single enzyme and other species can be studied with respect to electron transfer, homodimer or heterodimer interactions, or for construction of biological-based chips for standardizing cytochrome P450 metabolism or for high-throughput screening of pharmaceutical agents. Previous studies have generally immobilized P450 enzymes in a matrix or on a surface. Here, we have attached CYP2C9 to gold substrates such that the resulting construct maintains the ability to bind and metabolize substrates in the presence of NADPH and cytochrome P450 reductase. The activity of these chips is directly dependent upon the linkers used to attach CYP2C9 and to the presence of key molecules in the active site during enzyme attachment. A novel method to detect substrate-enzyme binding, namely, superconducting quantum interference device (SQUID) magnetometry, was used to monitor the binding of substrates. Most significantly, conditions that allow measurable CYP2C9 metabolism to occur have been developed.     

Fluorescence detection of ligand binding to labeled cytochrome P450 BM3

The cytochrome P450 superfamily of monoxygenases are highly relevant for pharmaceutical, environmental and biocatalytical applications. The binding of a substrate to their catalytic site is usually detectable by UV-vis spectroscopy as a low-to-high spin state transition of the heme iron. However, the discovery of potential new substrates is limited by the fact that some compounds do not cause the typical spin-shift even if they are oxidised by P450 enzymes. Here we report a fluorescence-based method able to detect the binding of such substrates to the heme domain of cytochrome P450 BM3 from Bacillus megaterium. The protein was labeled with the fluorescent probe N,N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-Yl)-ethylenediamine (IANBD). Arachidonic and lauric acids are substrates of P450 BM3 and were used to validate the method, as their binding can be detected both by a spin-shift of the Soret peak from 419 to 397 nm and by the fluorescence change of the labelled protein. The fluorescence emission of the probe linked to the protein increased by a value corresponding to 121 ± 9% and 52 ± 5% with respect to the initial one, upon titration with arachidonic or lauric acids respectively. The dissociation constants were calculated by both UV-vis and fluorescence spectroscopy. Three drugs, propranolol, chlorzoxazone and nifedipine, known to be oxidized by P450 BM3 and that bind without causing spin-shift, were also tested and the fluorescence emission of IANBD was found to decrease by 29 ± 5%, 21 ± 2% and 23 ± 3%, respectively, allowing the measurement of their dissociation constants.     

Oxygen activation by cytochrome p450: a thermodynamic analysis

Electrode potentials for every intermediate in the cytochrome P450 cycle were estimated and evaluated by means of an oxidation state diagram. By this approach, and within the uncertainties of the approximations, the superoxide complex of cytochrome P450 at pH 7 is oxidizing: E degrees ' (P450FeO(2)2+, H+/P450FeOOH2+) = +0.93 V, and the Gibbs energy for the reaction of the hydroperoxo complex of cytochrome P450 to form compound I and water, P450FeOOH2+ + H+ = P450FeO2+ por(*+) + H2O, is 0 kJ/mol. Although cytochrome P450FeOOH2+ and cytochrome P450FeO2+ por(*+) are approximately isoenergetic, they are likely to react at different rates with substrates and may yield different products. Homolysis of the hydroperoxo complex of cytochrome P450 to compound II and the hydroxyl radical, P450FeOOH2+ = P450FeO2+ + HO(*), is unfavorable (DeltaG degrees ' = +92 kJ/mol), as is the dissociation into HOO- and cytochrome P450Fe3+ (+73 kJ/mol). It is shown that the sum of the Gibbs energy of association for cytochrome P450Fe3+ with the hydroperoxo anion and the Gibbs energy for the one-electron reduction of cytochrome P450FeOOH2+, relative to NHE, is constant (-203 kJ/mol). While the estimated E degrees ' (P450FeO(2)2+, H+/P450FeOOH2+) = +0.93 V at pH 7 is larger than necessary to effect reduction of cytochrome P450FeO(2)2+, the magnitude of this electrode potential implies that the binding constant for cytochrome P450Fe3+ with hydrogen peroxide is ca. 3 x 106 M(-1) at pH 7. An association constant of this magnitude ensures that a fraction of cytochrome P450FeOOH2+ is available to form compound I or to react with substrates directly, while a larger one would imply that compound I is too weak an oxidant. In general, the energetics of the reduction of dioxygen to water determines the energetics of catalysis of hydroxylations by cytochrome P450. These results enable calibration of energy levels obtained for intermediates in the cytochrome P450 reaction cycle obtained by ab initio calculations and provide insights into the catalytic efficiency of cytochrome P450 and guidelines for the development of competent hydroxylation catalysts.     

Thermal equilibrium of high- and low-spin forms of cytochrome P450 BM-3: repositioning of the substrate?

We demonstrate that cytochrome P450 BM-3 in complex with N-palmitoylglycine undergoes a spin state change between room temperature, where optimal activity is seen, and low temperatures, where X-ray diffraction characterization has been carried out. On the basis of NMR measurements of the full-length protein, this spin state change is likely to be accompanied by a general structural rearrangement in the enzyme pocket. The substrate remains bound at all temperatures. We propose that the substrate may "slide" from a position directly atop the heme (thus displacing the ligating water) to the more distant position (thus restoring the ligating water) as the temperature is lowered. This proposal is evaluated on the basis of computational modeling of the protein-ligand complex, using a novel induced fit methodology. We thereby generate a structure with the ligand in close contact with the heme, similar in energy to the experimental structure. With this combination of theory and experiment we provide a specific proposal of how ligands may be positioned for chemistry for this enzyme.     

Crystals in Minutes: Instant On‐Site Microcrystallisation of Various Flavours of the CYP102A1 (P450BM3) Haem Domain

Despite CYP102A1 (P450BM3) representing one of the most extensively researched metalloenzymes, crystallisation of its haem domain upon modification can be a challenge. Crystal structures are indispensable for the efficient structure‐based design of P450BM3 as a biocatalyst. The abietane diterpenoid derivative N‐abietoyl‐l ‐tryptophan (AbiATrp) is an outstanding crystallisation accelerator for the wild‐type P450BM3 haem domain, with visible crystals forming within 2 hours and diffracting to a near‐atomic resolution of 1.22 Å. Using these crystals as seeds in a cross‐microseeding approach, an assortment of P450BM3 haem domain crystal structures, containing previously uncrystallisable decoy molecules and diverse artificial metalloporphyrins binding various ligand molecules, as well as heavily tagged haem‐domain variants, could be determined. Some of the structures reported herein could be used as models of different stages of the P450BM3 catalytic cycle.     

Electronic ground states of iron porphyrin and of the first species in the catalytic reaction cycle of cytochrome P450s

Electronic structures of iron(II) and iron(III) porphyrins are studied with density functional theory (DFT) using the GGA exchange functional OPTX in combination with the correlation functional PBE (OPBE) and with the correlation functional Perdew (OPerdew) together with a triple zeta-type basis set. These functionals, known for accurately predicting the spin ground state of iron complexes, are evaluated against other functionals for their performance in calculating relative energies for the various electronic states of both the iron porphyrins. The calculated energy orderings are triplet < quintet < singlet for the iron(II) porphyrin and quartet < sextet < doublet for the iron(III) porphyrin cation. Complexation by a thiolate ion (SH-) changes the preferred ground state for both species to high spin. This thiolate complex is used as a mimic for the cytochrome P450s active site to model the first step of the catalytic cycle of this enzyme. This first step is believed to concern the removal of an axial oxygen donating ligand from the hexacoordinated aqua-thiolate-porphyrin-iron(III) resting state. The DFT results suggest that this is not a free water molecule, because of its repulsive nature, but that it has instead hydroxy anion character. These calculations are in line with the experimentally observed change in the spin state from low to high spin upon this removal of the axial hydroxo ligand by binding of the substrate in the heme pocket of cytochrome P450.