Tailored nanodisc immobilization for one-step purification and reconstitution of cytochrome P450: A tool for membrane proteins' hard cases.

A novel nanodisc-based immobilization method was developed for high-efficient purification and reconstitution of cytochrome P450 in one step. Using membrane scaffold protein containing a histidine tag, charged-nanodiscs were prepared in the form of self-assembly of lipid-protein nanoparticles. Their properties including the particle diameter and its distribution and Zeta potential were controlled well by adjusting molar ratios of phospholipids to membrane scaffold protein. At an optimum lipid-to-membrane scaffold protein molar ratio of 60:1, uniformly regular-shaped and discoidal nanodiscs with an average particle diameter of 10 nm and Zeta potential of -19 mV were obtained. They can be well fractionated by size exclusion chromatography. Charged-nanodiscs were successfully immobilized onto Ni-chelating microspheres via histidine tags with a density of 6.6 mg membrane scaffold protein/mL gel. After being packed in a column, chromatography studies demonstrated that this nanodisc-immobilized chromatographic medium had a specific binding to cytochrome P450 in rat liver microsome. Nanodiscs containing cytochrome P450 can be furthermore eluted from the column with a diameter of about 87.0 nm and height of about 8.0 nm, respectively. The purity of cytochrome P450 after purification increased 25 folds strikingly. This nanodisc-immobilized chromatography method is promising for the one-step purification and reconstitution of membrane protein.

Detection of the First Epoxyalcohol Synthase/Allene Oxide Synthase (CYP74 Clan) in the Lancelet (Branchiostoma belcheri, Chordata).

The CYP74 clan cytochromes (P450) are key enzymes of oxidative metabolism of polyunsaturated fatty acids in plants, some Proteobacteria, brown and green algae, and Metazoa. The CYP74 enzymes, including the allene oxide synthases (AOSs), hydroperoxide lyases, divinyl ether synthases, and epoxyalcohol synthases (EASs) transform the fatty acid hydroperoxides to bioactive oxylipins. A novel CYP74 clan enzyme CYP440A18 of the Asian (Belcher's) lancelet (Branchiostoma belcheri, Chordata) was biochemically characterized in the present work. The recombinant CYP440A18 enzyme was active towards all substrates used: linoleate and α-linolenate 9- and 13-hydroperoxides, as well as with eicosatetraenoate and eicosapentaenoate 15-hydroperoxides. The enzyme specifically converted α-linolenate 13-hydroperoxide (13-HPOT) to the oxiranyl carbinol (9Z,11R,12R,13S,15Z)-11-hydroxy-12,13-epoxy-9,15-octadecadienoic acid (EAS product), α-ketol, 12-oxo-13-hydroxy-9,15-octadecadienoic acid (AOS product), and cis-12-oxo-10,15-phytodienoic acid (AOS product) at a ratio of around 35:5:1. Other hydroperoxides were converted by this enzyme to the analogous products. In contrast to other substrates, the 13-HPOT and 15-HPEPE yielded higher proportions of α-ketols, as well as the small amounts of cyclopentenones, cis-12-oxo-10,15-phytodienoic acid and its higher homologue, dihomo-cis-12-oxo-3,6,10,15-phytotetraenoic acid, respectively. Thus, the CYP440A18 enzyme exhibited dual EAS/AOS activity. The obtained results allowed us to ascribe a name "B. belcheri EAS/AOS" (BbEAS/AOS) to this enzyme. BbEAS/AOS is a first CYP74 clan enzyme of Chordata species possessing AOS activity.

Biased cytochrome P450-mediated metabolism via small-molecule ligands binding P450 oxidoreductase.

Metabolic control is mediated by the dynamic assemblies and function of multiple redox enzymes. A key element in these assemblies, the P450 oxidoreductase (POR), donates electrons and selectively activates numerous (>50 in humans and >300 in plants) cytochromes P450 (CYPs) controlling metabolism of drugs, steroids and xenobiotics in humans and natural product biosynthesis in plants. The mechanisms underlying POR-mediated CYP metabolism remain poorly understood and to date no ligand binding has been described to regulate the specificity of POR. Here, using a combination of computational modeling and functional assays, we identify ligands that dock on POR and bias its specificity towards CYP redox partners, across mammal and plant kingdom. Single molecule FRET studies reveal ligand binding to alter POR conformational sampling, which results in biased activation of metabolic cascades in whole cell assays. We propose the model of biased metabolism, a mechanism akin to biased signaling of GPCRs, where ligand binding on POR stabilizes different conformational states that are linked to distinct metabolic outcomes. Biased metabolism may allow designing pathway-specific therapeutics or personalized food suppressing undesired, disease-related, metabolic pathways.

Electrochemical and spectroscopic characteristics of cytochrome P450 55A3 and its interaction with nitric oxide.

Cytochrome P450 55A3 (CYP55A3) is an enzyme with the catalytic activity of nitric oxide (NO) to nitrous oxide using NADH or NADPH as the electron donor. Herein CYP55A3 has been expressed in E. coli and purified by His-tag columns. The electrochemical and spectroscopic characteristic of CYP55A3 and its interaction with NO has been studied. The direct electrochemistry of Fe3+/Fe2+ redox peaks in CYP55A3 was realized on the pyrolitic graphite electrode with the redox potential of -475 mV in pH 7.0 phosphate buffer. With the addition of NO a ferric nitroxyl complex (Fe3+-NO) formed with a new reduction peak at -0.78 V. The reduction peak current increased with the concentration of NO and showed typical Michaelis-Menten kinetic characteristics with the apparent Michaelis constant Kmapp 9.78 µM. The binding constant K calculated to be 3.93 × 104 M by UV-vis method. The fluorescence emission spectra of iron porphyrin in CYP55A3 showed with the peak wavelength 633 nm, and its fluorescence intensity increased after binding with NO. The fluorescence analysis demonstrated that NADH can relay electrons to iron porphyrin and reduce NO. The reductive product of NO released and the iron porphyrin in CYP55A3 turned back to the original form.

Characterization of a thermophilic cytochrome P450 of the CYP203A subfamily from Binh Chau hot spring in Vietnam.

Cytochromes P450 (CYPs or P450s) comprise a superfamily of heme-containing monooxygenases that are involved in a variety of biological processes. CYPs have broad utilities in industry, but most exhibit low thermostability, limiting their use on an industrial scale. Highly thermostable enzymes can be obtained from thermophiles in geothermal areas, including hot springs, offshore oil-producing wells and volcanoes. Here, we report the identification of a gene encoding for a thermophilic CYP from the Binh Chau hot spring metagenomic database, which was designated as P450-T2. The deduced amino acid sequence showed the highest identity of 73.15% with CYP203A1 of Rhodopseudomonas palustris, supporting that P450-T2 is a member of the CYP203A subfamily. Recombinant protein expression yielded 541 nm. The optimal temperature and pH of P450-T2 were 50 °C and 8.0, respectively. The half-life of P450-T2 was 50.2 min at 50 °C, and its melting temperature was 56.80 ± 0.08 °C. It was found to accept electrons from all tested redox partners systems, with BmCPR-Fdx2 being the most effective partner. Screening for putative substrates revealed binding of phenolic compounds, such as l-mimosine and emodin, suggesting a potential application of this new thermophilic P450 in the production of the corresponding hydroxylated products.

Double Electron-Electron Resonance Shows That the Substrate but Not the Inhibitors Causes Disorder in the F/G Loop of CYP119 in Solution.

CYP119, a bacterial thermophilic protein from the cytochrome P450 superfamily, has previously been observed in three different conformations with different inhibitors bound using X-ray crystallography. The significance of these states in solution and in the function of the enzyme is not well-known. Double electron-electron resonance (DEER) was used to measure distances and distance distributions between spin-labels for populated conformational states in solution. DEER spectroscopy and molecular dynamics for the ligand-free enzyme suggest that the G helix is in a slightly different conformation than seen previously by crystallography, with the F/G loop in a slightly open conformation. Inhibitor-bound samples showed that this conformation remains as the predominant form, but partial conversion is indicated to a more closed conformation of the F/G loop. However, when the enzyme binds to lauric acid, the proposed substrate, it induces the conversion to a state that is characterized by increased disorder. We propose that similar to recent results with soluble CYP3A4, binding of the inhibitor to CYP119 is accompanied by only small changes in the enzyme structure, but substrate binding results in greater heterogeneity in the structure of the F/G loop region.

Oxygen Surrogate Systems for Supporting Human Drug-Metabolizing Cytochrome P450 Enzymes.

Oxygen surrogates (OSs) have been used to support cytochrome P450 (P450) enzymes for diverse purposes in drug metabolism research, including reaction phenotyping, mechanistic and inhibition studies, studies of redox partner interactions, and to avoid the need for NADPH or a redox partner. They also have been used in engineering P450s for more cost-effective, NADPH-independent biocatalysis. However, despite their broad application, little is known of the preference of individual P450s for different OSs or the substrate dependence of OS-supported activity. Furthermore, the biocatalytic potential of OSs other than cumene hydroperoxide (CuOOH) and hydrogen peroxide (H2O2) is yet to be explored. Here, we investigated the ability of the major human drug-metabolizing P450s, namely CYP3A4, CYP2C9, CYP2C19, CYP2D6, and CYP1A2, to use the following OSs: H2O2, tert-butyl hydroperoxide (tert-BuOOH), CuOOH, (diacetoxyiodo)benzene, and bis(trifluoroacetoxy)iodobenzene. Overall, CuOOH and tert-BuOOH were found to be the most effective at supporting these P450s. However, the ability of P450s to be supported by OSs effectively was also found to be highly dependent on the substrate used. This suggests that the choice of OS should be tailored to both the P450 and the substrate under investigation, underscoring the need to employ screening methods that reflect the activity toward the substrate of interest to the end application. SIGNIFICANCE STATEMENT: Cytochrome P450 (P450) enzymes can be supported by different oxygen surrogates (OSs), avoiding the need for a redox partner and costly NADPH. However, few data exist comparing relative activity with different OSs and substrates. This study shows that the choice of OS used to support the major drug-metabolizing P450s influences their relative activity and regioselectivity in a substrate-specific fashion and provides a model for the more efficient use of P450s for metabolite biosynthesis.

Peroxide-driven catalysis of the heme domain of A. radioresistens cytochrome P450 116B5 for sustainable aromatic rings oxidation and drug metabolites production.

The heme domain of cytochrome P450 116B5 from Acinetobacter radioresistens (P450 116B5hd), a self-sufficient class VII P450, was functionally expressed in Escherichia coli, purified and characterised in active form. Its unusually high reduction potential (-144 ±â€¯42 mV) and stability in the presence of hydrogen peroxide make this enzyme a good candidate for driving catalysis with the so-called peroxide shunt, avoiding the need for a reductase and the expensive cofactor NAD(P)H. The enzyme is able to carry out the peroxide-driven hydroxylation of aromatic compounds such as p-nitrophenol (KM = 128.85 ±â€¯29.51 µM and kcat = 2.65 ±â€¯0.14 min-1), 10-acetyl-3,7-dihydroxyphenoxazine (KM = 6.01 ±â€¯0.32 µM and kcat = 0.33 ±â€¯0.03 min-1), and 3,5,3',5'tetramethylbenzidine (TMB). Moreover, it catalyses different reactions on well-known drugs such as hydroxylation of diclofenac (KM = 49.60 ±â€¯6.30 µM and kcat = 0.06 ±â€¯0.01 min-1) and N-desmethylation of tamoxifen (KM = 57.20 ±â€¯7.90 µM and kcat = 0.79 ±â€¯0.04 min-1). The data demonstrate that P450 116B5hd is an efficient biocatalyst for sustainable applications in bioremediation and human drug metabolite production.

Regio- and stereoselective hydroxylation of testosterone by a novel cytochrome P450 154C2 from Streptomyces avermitilis.

Cytochrome P450 enzymes (P450 or CYP) are some of the most versatile biocatalysts, and offer advantages for oxidizing unreactive C-H bonds in mild conditions. In this study, we identified a novel cytochrome P450 154C2 from Streptomyces avermitilis and characterized its function in 2α-hydroxylation of testosterone with regio- and stereoselectivity. To investigate the efficiency of electron transfer, we conducted biotransformation using two different P450 redox partners-RhFRED (RhF reductase domain) from Rhodococcus sp. and Pdx (putidaredoxin)/Pdr (putidaredoxin reductase) from Pseudomonas putida and revealed that RhFRED was more effective than Pdx/Pdr, especially in vivo. The Km and kcat values for testosterone were estimated to be 0.16 ± 0.05 mM and 0.13 ± 0.02 min-1, and kcat/Km was 0.81 min-1 mM-1. We also determined the crystal structure of the substrate-free form of CYP154C2 at 1.5 Å resolution. The structure has a closed conformation, and the substrate binding pocket is narrow, which can explain the strict substrate specificity of the enzyme.

Evaluation of Luminogenic Substrates as Probe Substrates for Bacterial Cytochrome P450 Enzymes: Application to Mycobacterium tuberculosis.

Several cytochrome P450 enzymes (CYPs) encoded in the genome of Mycobacterium tuberculosis (Mtb) are considered potential new drug targets due to the essential roles they play in bacterial viability and in the establishment of chronic intracellular infection. Identification of inhibitors of Mtb CYPs at present is conducted by ultraviolet-visible (UV-vis) optical titration experiments or by metabolism studies using endogenous substrates, such as cholesterol and lanosterol. The first technique requires high enzyme concentrations and volumes, while analysis of steroid hydroxylation is dependent on low-throughput analytical methods. Luciferin-based luminogenic substrates have proven to be very sensitive substrates for the high-throughput profiling of inhibitors of human CYPs. In the present study, 17 pro-luciferins were evaluated as substrates for Mtb CYP121A1, CYP124A1, CYP125A1, CYP130A1, and CYP142A1. Luciferin-BE was identified as an excellent probe substrate for CYP130A1, resulting in a high luminescence yield after addition of luciferase and adenosine triphosphate (ATP). Its applicability for high-throughput screening was supported by a high Z'-factor and high signal-to-background ratio. Using this substrate, the inhibitory properties of a selection of known inhibitors could be characterized using significantly less protein concentration when compared to UV-vis optical titration experiments. Although several luminogenic substrates were also identified for CYP121A1, CYP124A1, CYP125A1, and CYP142A1, their relatively low yield of luminescence and low signal-to-background ratios make them less suitable for high-throughput screening since high enzyme concentrations will be needed. Further structural optimization of luminogenic substrates will be necessary to obtain more sensitive probe substrates for these Mtb CYPs.

CYP52A23 from Candida albicans and its Substrate Preference for Fatty Acid Hydroxylation.

The pathogenic fungus Candida albicans contains genes encoding five fatty acid hydroxylases belonging to the CYP52 family in its genome. Our previous study reported that CYP52A21 demonstrated typical omega-hydroxylation of lauric acid (Kim D, Cryle MJ, De Voss JJ, Ortiz de Montellano PR (2007) Arch Biochem Biophys 464, 213-220). Functional characterization of CYP52 fatty acid hydroxylases was studied, and their selectivity for hydroxylation was analyzed. Genes for four other CYP52 members (CYP52A22, CYP52A23, CYP52A24, and CYP52C3) from C. albicans were cloned, and their recombinant enzymes were expressed in Escherichia coli. CO-binding spectral analyses showed that the functional P450 holoenzyme was obtained only in CYP52A23, while no holoenzyme peak was observed in the other three CYP52 enzymes. Spectral change of the type II binding was observed in purified CYP52A23 when titrated with fatty acids but none was observed with alkanes. The gas chromatography-mass spectrometry (GC-MS) analysis revealed that CYP52A23 predominantly exhibited omega-hydroxylation activity during the oxidation reaction of fatty acids. Interestingly, it was found that CYP52A23 preferred longer-chain fatty acids (stearic acid and arachidic acid) for its catalytic activities while CYP52A21 preferred mid-chain fatty acids (lauric acid and mystic acid). To analyze the selectivity of fatty acids, hybrid mutagenesis of genes encoding CYP52A21 and CYP52A23 by overlap extension polymerase chain reaction was conducted. Two hybrid mutants containing the N-terminal fragments of CYP52A21 and C-terminal fragments of CYP52A23 displayed higher catalytic activity in palmitic acid and arachidic acid. These results suggested that the C-terminal part of CYP52A23 may be responsible for its preference to longer-chain fatty acids.

Analysis and preliminary characterisation of the cytochrome P450 monooxygenases from Frankia sp. EuI1c (Frankia inefficax sp.).

Frankia bacteria are nitrogen fixing species from the Actinobacterium phylum which live on the root nodules of plants. They have been hypothesised to have significant potential for natural product biosynthesis. The cytochrome P450 monooxygenase complement of Frankia sp. EuI1c (Frankia inefficax sp.), which comprises 68 members, was analysed. Several members belonged to previously uncharacterised bacterial P450 families. There was an unusually high number of CYP189 family members (21) suggesting that this family has undergone gene duplication events which are classified as "blooms". The likely electron transfer partners for the P450 enzymes were also identified and analysed. These consisted of predominantly [3Fe-4S] cluster containing ferredoxins (eight), a single [2Fe-2S] ferredoxin and a couple of ferredoxin reductases. Three of these CYP family members were produced and purified, using Escherichia coli as a host, and their substrate range was characterised. CYP1027H1 and CYP150A20 bound a broad range of norisoprenoids and terpenoids. CYP1074A2 was highly specific for certain steroids including testosterone, progesterone, stanolone and 4-androstene-3,17-dione. It is likely that steroids are the physiological substrates of CYP1074A2. These results also give an indication that terpenoids are the likely substrates of CYP1027H1 and CYP150A2. The large number of P450s belonging to distinct families as well as the associated electron transfer partners found in different Frankia strains highlights the importance of this family of enzymes has in the secondary metabolism of these bacteria.

Development and validation of an absolute protein assay for the simultaneous quantification of fourteen CYP450s in human microsomes by HPLC-MS/MS-based targeted proteomics.

The cytochrome P450 (CYP450) superfamily constitutes the major enzymatic system involved in drug metabolism. CYP450s are highly expressed in the liver and other tissues and limited data on absolute characterization of CYP450s in extra hepatic organs, such as the small intestine, are available. Our objective was to develop and validate an absolute quantification assay by HPLC-MS/MS-based targeted proteomics allowing the simultaneous quantification of fourteen major human CYP450 isoforms (CYP1A1, 1A2, 1B1, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 2J2, 3A4, 3A5, 3A7 and 4F2) in human liver and intestine microsomes. Absolute protein quantification was performed using two proteotypic peptides for each of the fourteen CYP450s. Peptides were obtained after a tryptic digestion of microsomes and samples were analyzed by high performance liquid chromatography with heated electrospray ionization tandem mass spectrometry (HPLC-HESI-MS/MS). Chromatographic separation was performed on a Biobasic-8 analytical column (5 µm 100 x 1 mm) with a gradient elution using acetonitrile and water both fortified with 0.1% formic acid (flow rate: 75 µL/min). Calibration curves were linear over a wide range of concentrations (0.1-50 nM) and the assay met all requirements of sensitivity, linearity, precision, accuracy and matrix effect. Strong correlations were observed between the two proteotypic peptides for each isoenzyme, corroborating the strength of this method. Twelve CYP450s were detected in commercially available human liver microsomes while seven CYP450s were detected in human intestine microsomes. To our knowledge, this is the most sensitive (0.1 nM) and the first most extensively validated assay that can be applied to the absolute quantification of CYP450s in human tissues.

The characterisation of two members of the cytochrome P450 CYP150 family: CYP150A5 and CYP150A6 from Mycobacterium marinum.

BACKGROUND: Actinobacteria, including the Mycobacteria, have a large component of cytochrome P450 family monooxygenases. This includes Mycobacterium tuberculosis, M. ulcerans and M. marinum, and M. vanbaalenii. These enzymes can abstract CH bonds and have important roles in natural product biosynthesis. METHODS: Two members of the bacterial CYP150 family, CYP150A5 and CYP150A6 from M. marinum, were produced, purified and characterised. The potential substrate ranges of both enzymes were analysed and the monooxygenase activity of CYP150A5 was reconstituted using a physiological electron transfer partner system. CYP150A6 was structurally characterised by X-ray crystallography. RESULTS: CYP150A5 was shown to bind various norisoprenoids and terpenoids. It could regioselectively hydroxylate ß-ionol. The X-ray crystal structure of substrate-free CYP150A6 was solved to 1.5 Å. This displayed an open conformation with short F and G helices, an unresolved F-G loop region and exposed active site pocket. The active site residues could be identified and important variations were found among the CYP150A enzymes. Haem-binding azole inhibitors were identified for both enzymes. CONCLUSIONS: The structure of CYP150A6 will facilitate the identification of physiological substrates and the design of better inhibitors for members of this P450 family. Based on the observed differences in substrate binding preference and sequence variations among the active site residues, their roles are predicted to be different. GENERAL SIGNIFICANCE: Multiple CYP150 family members were found in many bacteria and are prevalent in the Mycobacteria including several human pathogens. Inhibition and structural data are reported here for these enzymes for the first time.

Bacterial Expression of Membrane-Associated Cytochrome P450s and Their Activity Assay in Nanodiscs.

Eukaryotic membrane bound cytochrome P450s are expressed in bacterial systems to produce large yields of catalytically active protein for structure function studies. Recently, there have been several instances of expressing eukaryotic membrane bound CYPs in bacteria after making various modifications to both the N-terminus membrane binding domains of the protein and to noncontiguous F-G membrane binding loop that is also implicated in substrate binding. These modifications have been shown not to disturb the function of the protein of interest. The major factors that have been key to express the membrane bound cytochrome P450s in bacteria have been the following: (a) exon optimization (b) selection of the appropriate vector and host strain, and (c) growth and expression conditions with respect to temperature and speed of shaking the media flask. Herein, we describe methods to express and purify eukaryotic membrane bound cytochrome P450s. We also describe the measurement of the activity of the cytochrome P450 expressed by taking the example of cytochrome P450 2J2, the primary P450 expressed in the human heart and CYP725A4, the primary cytochrome P450 expressed in the first step of taxol synthesis. Additionally, we discuss the pros and cons of the different modifications done in order to express the membrane bound cytochrome P450s.

Molecular snapshots of dynamic membrane-bound metabolons.

Numerous biosynthetic pathways have been shown to assemble at the surface of cellular membranes into efficient dynamic supramolecular assemblies termed metabolons. In response to environmental stimuli, metabolons assemble on-demand making them highly dynamic and fragile. This transient nature has previously hampered isolation and molecular characterization of dynamic metabolons. In contrast to conventional detergents, which tend to disrupt weak protein-protein interactions and remove lipids, the competence of a styrene maleic acid copolymer to carve out discrete lipid nanodisc from membranes offers immense potential for isolation of intact protein assemblies. Here, we present a method to extract the entire membrane-bound dhurrin pathway directly from microsomal fractions of the cereal Sorghum bicolor. This detergent-free nanodisc approach may be generally transposed for isolation of entire plant biosynthetic metabolons. This method provides a simple practical toolkit for the study of membrane protein complexes.

Novel Point Mutations in cyp51A and cyp51B Genes Associated with Itraconazole and Posaconazole Resistance in Aspergillus clavatus Isolates.

Aspergillus clavatus is a common environmental species known to cause occupational allergic disease in grain handlers. We have recently observed azole-resistant isolates of this fungus as a cause of onychomycosis. To further characterize the cause of resistance, the genes encoding 14 α-sterol demethylase enzyme (cyp51A and cyp51B) were characterized and analyzed in 9 ITC-susceptible isolates and 6 isolates with high minimum inhibitory concentrations (MICs) of clinical (nail and sputum) and environmental A. clavatus strains. We found that six isolates with itraconazole MIC >16 mg/L demonstrated nonsynonymous mutations, including V51I, L378P, E483K, and E506G, and synonymous mutations, including F53F, A186A, Q276Q, and H359H. Moreover, P486S was detected in five strains with ITR MIC >16 mg/L. One mutation, F324S, was detected in an isolate with posaconazole MIC >16 mg/L. The effect of E483K and P486S mutations of CYP51A on azole resistance was further investigated using homology modeling and molecular dynamics. We found that E483K and P486S mutations were located near the ligand access channel of CYP51A that could partly lead to narrowing the entry of the ligand access channels. Therefore, we concluded that E483K and P486S mutations may potentially contribute to the limited access of inhibitors to the binding pocket and therefore confer resistance to azole agents.

Characterization of Two Self-Sufficient Monooxygenases, CYP102A15 and CYP102A170, as Long-Chain Fatty Acid Hydroxylases.

Self-sufficient P450s, due to their fused nature, are the most effective tools for electron transfer to activate C-H bonds. They catalyze the oxygenation of fatty acids at different omega positions. Here, two new, self-sufficient cytochrome P450s, named CYP102A15 and CYP102A170, from polar Bacillus sp. PAMC 25034 and Paenibacillus sp. PAMC 22724, respectively, were cloned and expressed in E. coli. The genes are homologues of CYP102A1 from Bacillus megaterium. They catalyzed the hydroxylation of both saturated and unsaturated fatty acids ranging in length from C12-C20, with a moderately diverse profile compared to other members of the CYP102A subfamily. CYP102A15 exhibited the highest activity toward linoleic acid with Km 15.3 µM, and CYP102A170 showed higher activity toward myristic acid with Km 17.4 µM. CYP10A170 also hydroxylated the Eicosapentaenoic acid at ω-1 position only. Various kinetic parameters of both monooxygenases were also determined.

Detection of the first higher plant epoxyalcohol synthase: Molecular cloning and characterisation of the CYP74M2 enzyme of spikemoss Selaginella moellendorffii.

The CYP74M2 gene of a model plant, the spikemoss Selaginella moellendorffii Hieron, was cloned and the catalytic properties of corresponding recombinant protein were studied. The recombinant CYP74M2 protein was active towards 13-hydroperoxides of linoleic and a-linolenic acids (13-HPOD and 13-HPOT, respectively). In contrast to previously studied CYP74M1 and CYP74M3, which possessed the divinyl ether synthase activity, CYP74M2 behaved as a dedicated epoxyalcohol synthase (EAS). For instance, the 13-HPOD was converted to three epimeric oxiranyl carbinols 1-3 (formed at a ratio ca. 4:2:1), namely the (11R,12S,13S), (11R,12R, 13S), and (11S,12S,13S) epimers of (9Z)-11-hydroxy-12,13-epoxy-9-octadecenoic acid. Besides these products, a minority of oxiranyl vinyl carbinols like (10E)-11-hydroxy-12,13-epoxy-9-octadecenoic acid was formed. The 13-HPOT conversion by CYP74M2 afforded two stereoisomers of 11-hydroxy-12,13-epoxy-9,15-octadecadienoic acid. Individual oxylipins were purified by HPLC and finally identified by their NMR data, including the 1H-NMR, 2D-COSY, HSQC, and HMBC. Thus, the CYP74M2 is the dedicated epoxyalcohol synthase. To our knowledge, no enzymes of this type have been detected in higher plants yet.

Characterization of Cytochrome P450 Enzymes and Their Applications in Synthetic Biology.

The cytochrome P450 monooxygenase enzymes (P450s) catalyze a diverse array of chemical transformations, most originating from the insertion of an oxygen atom into a substrate that binds close to the P450 heme. The oxygen is delivered by a highly reactive heme iron-oxo species (compound I) and, according to the chemical nature of the substrate and its position in the active site, the P450 can catalyze a wide range of reactions including, e.g., hydroxylation, reduction, decarboxylation, sulfoxidation, N- and O-demethylation, epoxidation, deamination, CC bond formation and breakage, nitration, and dehalogenation. In this chapter, we describe the structural, biochemical, and catalytic properties of the P450s, along with spectroscopic and analytical methods used to characterize P450 enzymes and their redox partners. Important uses of P450 enzymes are highlighted, including how various P450s have been exploited for applications in synthetic biology.