Structural determination and characterisation of the CYP105Q4 cytochrome P450 enzyme from Mycobacterium marinum.

The cytochrome P450 family of heme metalloenzymes (CYPs) catalyse important biological monooxygenation reactions. Mycobacterium marinum contains a gene encoding a CYP105Q4 enzyme of unknown function. Other members of the CYP105 CYP family have key roles in bacterial metabolism including the synthesis of secondary metabolites. We produced and purified the cytochrome P450 enzyme CYP105Q4 to enable its characterization. Several nitrogen-donor atom-containing ligands were found to bind to CYP105Q4 generating type II changes in the UV-vis absorbance spectrum. Based on the UV-vis absorbance spectra none of the potential substrate ligands we tested with CYP105Q4 were able to displace the sixth distal aqua ligand from the heme, though there was evidence for binding of oleic acid and amphotericin B. The crystal structure of CYP105Q4 in the substrate-free form was determined in an open conformation. A computational structural similarity search (Dali) was used to find the most closely related characterized relatives within the CYP105 family. The structure of CYP105Q4 enzyme was compared to the GfsF CYP enzyme from Streptomyces graminofaciens which is involved in the biosynthesis of a macrolide polyketide. This structural comparison to GfsF revealed conformational changes in the helices and loops near the entrance to the substrate access channel. A disordered B/C loop region, usually involved in substrate recognition, was also observed.

Selective carbon-hydrogen bond hydroxylation using an engineered cytochrome P450 peroxygenase.

The cytochrome P450 enzyme CYP102A1 (P450BM3) is a versatile monooxygenase enzyme which has been adapted and engineered for multiple applications in chemical synthesis. Mutation of threonine 268 to glutamate (Thr268Glu) converted the heme domain of this enzyme into a H2O2 utilizing peroxygenase. This variant displayed significantly increased peroxide driven hydroxylation activity towards the saturated linear fatty acids tested (undecanoic through to hexadecenoic acid) when compared to the wild-type heme domain. The product distributions arising from fatty acid oxidation using this peroxygenase variant were broadly similar to those obtained with the wild-type monooxygenase holoenzyme, with oxidation occurring predominantly at the ω-1 through to ω-3 positions. 10-Undecenoic acid was regioselectively hydroxylated at the allylic ω-2 carbon by the Thr268Glu peroxygenase. The effect of isotopic substitution were measured using [9,9,10,10-d4]-dodecanoic acid. The kinetic isotope effect for both the monooxygenase and peroxygenase systems ranged between 7.9 and 9.5, with that of the peroxygenase enzyme being marginally lower. This highlights that carbon­hydrogen bond abstraction is important in the mechanism of both the monooxygenase and peroxygenase systems. This would infer that the ferryl-oxo radical cation intermediate, compound I, is the likely reactive intermediate in both systems. The peroxygenase variant offers the possibility of simpler cytochrome P450 systems for selective oxidations. To demonstrate this we used this system to oxidize tetradecanoic acid using light driven generation of H2O2 by a flavin.

A comparison of the bacterial CYP51 cytochrome P450 enzymes from Mycobacterium marinum and Mycobacterium tuberculosis.

Members of the CYP51 family of cytochrome P450 enzymes are classified as sterol demethylases involved in the metabolic formation of cholesterol and related derivatives. The CYP51 enzyme from Mycobacterium marinum was studied and compared to its counterpart from Mycobacterium tuberculosis to determine the degree of functional conservation between them. Spectroscopic analyses of substrate and inhibitor binding of the purified CYP51 enzymes from M. marinum and M. tuberculosis were performed. The catalytic oxidation of lanosterol and related steroids was investigated. M. marinum CYP51 was structurally characterized by X-ray crystallography. The CYP51 enzyme of M. marinum is sequentially closely related to CYP51B1 from M. tuberculosis. However, differences in the heme spin state of each enzyme were observed upon the addition of steroids and other ligands. Both enzymes displayed different binding properties to those reported for the CYP51-Fdx fusion protein from the bacterium Methylococcus capsulatus. The enzymes were able to oxidatively demethylate lanosterol to generate 14-demethylanosterol, but no products were detected for the related species dihydrolanosterol and eburicol. The crystal structure of CYP51 from M. marinum in the absence of added substrate but with a Bis-Tris molecule within the active site was resolved. The CYP51 enzyme of M. marinum displays differences in how steroids and other ligands bind compared to the M. tuberculosis enzyme. This was related to structural differences between the two enzymes. Overall, both of these CYP51 enzymes from mycobacterial species displayed significant differences to the CYP51 enzymes of eukaryotic species and the bacterial CYP51-Fdx enzyme of Me. capsulatus.

The Stereoselective Oxidation of para-Substituted Benzenes by a Cytochrome P450 Biocatalyst.

The serine 244 to aspartate (S244D) variant of the cytochrome P450 enzyme CYP199A4 was used to expand its substrate range beyond benzoic acids. Substrates, in which the carboxylate group of the benzoic acid moiety is replaced were oxidised with high activity by the S244D mutant (product formation rates >60 nmol.(nmol-CYP)-1 .min-1 ) and with total turnover numbers of up to 20,000. Ethyl α-hydroxylation was more rapid than methyl oxidation, styrene epoxidation and S-oxidation. The S244D mutant catalysed the ethyl hydroxylation, epoxidation and sulfoxidation reactions with an excess of one stereoisomer (in some instances up to >98 %). The crystal structure of 4-methoxybenzoic acid-bound CYP199A4 S244D showed that the active site architecture and the substrate orientation were similar to that of the WT enzyme. Overall, this work demonstrates that CYP199A4 can catalyse the stereoselective hydroxylation, epoxidation or sulfoxidation of substituted benzene substrates under mild conditions resulting in more sustainable transformations using this heme monooxygenase enzyme.

Kinetic Evidence for an Induced Fit Mechanism in the Binding of the Substrate Camphor by Cytochrome P450cam.

Bacterial cytochrome P450 (P450) 101A1 (P450cam) has served as a prototype among the P450 enzymes and has high catalytic activity towards its cognate substrate, camphor. X-ray crystallography and NMR and IR spectroscopy have demonstrated the existence of multiple conformations of many P450s, including P450cam. Kinetic studies have indicated that substrate binding to several P450s is dominated by a conformational selection process, in which the substrate binds an individual conformer(s) of the unliganded enzyme. P450cam was found to differ in that binding of the substrate camphor is dominated by an induced fit mechanism, in which the enzyme binds camphor and then changes conformation, as evidenced by the equivalence of binding eigenvalues observed when varying both camphor and P450cam concentrations. The accessory protein putidaredoxin had no effect on substrate binding. Estimation of the rate of dissociation of the P450cam·camphor complex (15 s-1) and fitting of the data yield a minimal kinetic mechanism in which camphor binds (1.5 × 107 M-1 s-1) and the initial P450cam•camphor complex undergoes a reversible equilibrium (k forward 112 s-1, k reverse 28 s-1) to a final complex. This induced fit mechanism differs from those reported for several mammalian P450s and bacterial P450BM-3, indicative of the diversity of how P450s recognize multiple substrates. However, similar behavior was not observed with the alternate substrates (+)-α-pinene and 2-adamantanone, which probably utilize a conformational selection process.

Characterization of human adrenal cytochrome P450 11B2 products of progesterone and androstenedione oxidation.

Cytochrome P450 (P450) 11B1 and 11B2 both catalyze the 11ß-hydroxylation of 11-deoxycorticosterone and the subsequent 18-hydroxylation of the product. P450 11B2, but not P450 11B1, catalyzes a further C-18 oxidation to yield aldosterone. 11-Oxygenated androgens are of interest, and 11-hydroxy progesterone has been reported to be a precursor of these. Oxidation of progesterone by purified recombinant P450 11B2 yielded a mono-hydroxy derivative as the major product, and co-chromatography with commercial standards and 2-D NMR spectroscopy indicated 11ß-hydroxylation. 18-Hydroxyprogesterone and a dihydroxyprogesterone were also formed. Similarly, oxidation of androstenedione by P450 11B2 yielded 11ß-hydroxyandrostenedione, 18-hydroxyandrostenedione, and a dihydroxyandrostenedione. The steady-state kinetic parameters for androstenedione and progesterone 11ß-hydroxylation were similar to those reported for the classic substrate 11-deoxycorticosterone. The source of 11α-hydroxyprogesterone in humans remains unresolved.

Functional interactions of adrenodoxin with several human mitochondrial cytochrome P450 enzymes.

Seven of the 57 human cytochrome P450 (P450) enzymes are mitochondrial and carry out important reactions with steroids and vitamins A and D. These seven P450s utilize an electron transport chain that includes NADPH, NADPH-adrenodoxin reductase (AdR), and adrenodoxin (Adx) instead of the diflavin NADPH-P450 reductase (POR) used by the other P450s in the endoplasmic reticulum. Although numerous studies have been published involving mitochondrial P450 systems, the experimental conditions vary considerably. We compared human Adx and bovine Adx, a commonly used component, and found very similar catalytic activities in reactions catalyzed by human P450s 11B2, 27A1, and 27C1. Binding constants of 6-200 nM were estimated for Adx binding to these P450s using microscale thermophoresis. All P450 catalytic reactions were saturated at 10 µM Adx, and higher concentrations were not inhibitory up to at least 50 µM. Collectively these studies demonstrate the tight binding of Adx (both human and bovine) to AdR and to several mitochondrial P450s and provide guidance for optimization of Adx-dependent P450 reactions.

A comparison of steroid and lipid binding cytochrome P450s from Mycobacterium marinum and Mycobacterium tuberculosis.

The steroid lipid binding cytochrome P450 (CYP) enzymes of Mycobacterium tuberculosis are essential for organism survival through metabolism of cholesterol and its derivatives. The counterparts to these enzymes from Mycobacterium marinum were studied to determine the degree of functional conservation between them. Spectroscopic analyses of substrate and inhibitor binding for the four M. marinum enzymes CYP125A6, CYP125A7, CYP142A3 and CYP124A1 were performed and compared to the equivalent enzymes of M. tuberculosis. The sequence of CYP125A7 from M. marinum was more similar to CYP125A1 from M. tuberculosis than CYP125A6 but both showed differences in the resting heme spin state and in the binding modes and affinities of certain azole inhibitors. CYP125A7 did not show a significant Type II inhibitor-like shift with any of the azoles tested. CYP142A3 bound a similar range of steroids and inhibitors to CYP142A1. However, there were some differences in the extent of the Type I shifts to the high-spin form with steroids and a higher affinity for the azole inhibitors compared to CYP142A1. The two CYP124 enzymes had similar substrate binding properties. M. marinum CYP124 was characterised by X-ray crystallography and displayed strong conservation of active site residues, except near the region where the carboxylate terminus of the phytanic acid substrate would be bound. As these enzymes in M. tuberculosis have been identified as candidates for inhibition the data here demonstrates that alternative strategies for inhibitor design may be required to target CYP family members from distinct pathogenic Mycobacterium species or other bacteria.

Multistep Binding of the Non-Steroidal Inhibitors Orteronel and Seviteronel to Human Cytochrome P450 17A1 and Relevance to Inhibition of Enzyme Activity.

Orteronel (TAK-700) is a substituted imidazole that was developed for the treatment of castration-resistant prostate cancer but was dropped in phase III clinical trials. Both enantiomers of this inhibitor of cytochrome P450 (P450) 17A1 show some selectivity in differentially blocking the 17α-hydroxylation and lyase activities of the enzyme. Although both enantiomers of this compound have sub-micromolar IC50 values and bind to the enzyme with a type II spectral change (indicative of nitrogen-iron bonding) and reported Kd values of 56 and 40 nM (R and S, respectively), the rates of binding to P450 17A1 were relatively slow. We considered the possibility that the drug is a slow, tight-binding inhibitor. Analysis of the kinetics of binding revealed rapid formation of an initial complex, presumably in the substrate binding site, followed by a slower change to the spectrum of a final iron complex. Similar kinetics were observed in the interaction of another inhibitor, the triazole (S)-seviteronel (VT-464), with P450 17A1. Kinetic tests and modeling indicate that the further change to the iron-complexed form of the orteronel- or seviteronel-P450 complex is not a prerequisite for enzyme inhibition. Accordingly, the inclusion of heme-binding heterocyclic nitrogen moieties in P450 17A1 inhibitors may not be necessary to achieve inhibition but may nevertheless augment the process.

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.

Selective ϖ-1 oxidation of fatty acids by CYP147G1 from Mycobacterium marinum.

BACKGROUND: Cyp147G1 is one of 47 cytochrome P450 encoding genes in Mycobacterium marinum M, a pathogenic bacterium with a high degree of sequence similarity to Mycobacterium tuberculosis and Mycobacterium ulcerans. Cyp147G1 is one of only two of these cyp genes which are closely associated with a complete electron transfer system. METHODS: The substrate range of the enzyme was tested in vitro and the activity of CYP147G1 was reconstituted in vivo by co-producing the P450 with the ferredoxin and ferredoxin reductase. RESULTS: Substrates of CYP147G1 include fatty acids ranging from octanoic to hexadecanoic acid. CYP147G1 catalysed the selective hydroxylation of linear and ω-2 methyl branched fatty acids at the ω-1 position (≥ 98%). Oxidation of ω-1 methyl branched fatty acids generated the ω and ω-1 hydroxylation products in almost equal proportions, indicating altered position of hydrogen abstraction. CONCLUSIONS: This selectivity of fatty acid hydroxylation inferred that linear species must bind in the active site of the enzyme with the terminal methyl group sequestered so that abstraction at the CH bonds of the ω-1 position is favoured. With branched substrates, one of the methyl groups must be close to the compound I oxygen atom and enable hydroxylation at the terminal methyl group to compete with the reaction at the ω-1CH bond. GENERAL SIGNIFICANCE: Hydroxy fatty acids are widely used for industrial, food and medical purposes. CYP147G1 demonstrates high regioselectivity for hydroxylation at a sub-terminal position on a broad range of linear fatty acids, not seen in other CYP enzymes.

Electron transfer ferredoxins with unusual cluster binding motifs support secondary metabolism in many bacteria.

The proteins responsible for controlling electron transfer in bacterial secondary metabolism are not always known or characterised. Here we demonstrate that many bacteria contain a set of unfamiliar ferredoxin encoding genes which are associated with those of cytochrome P450 (CYP) monooxygenases and as such are involved in anabolic and catabolic metabolism. The model organism Mycobacterium marinum M contains eleven of these genes which encode [3Fe-4S] or [4Fe-4S] single cluster containing ferredoxins but which have unusual iron-sulfur cluster binding motif sequences, CXX?XXC(X) n CP, where '?' indicates a variable amino acid residue. Rather than a cysteine residue, which is highly conserved in [4Fe-4S] clusters, or alanine or glycine residues, which are common in [3Fe-4S] ferredoxins, these genes encode at this position histidine, asparagine, tyrosine, serine, threonine or phenylalanine. We have purified, characterised and reconstituted the activity of several of these CYP/electron transfer partner systems and show that all those examined contain a [3Fe-4S] cluster. Furthermore, the ferredoxin used and the identity of the variable motif residue in these proteins affects the functionality of the monooxygenase system and has a significant influence on the redox properties of the ferredoxins. Similar ferredoxin encoding genes were identified across Mycobacterium species, including in the pathogenic M. tuberculosis and M. ulcerans, as well as in a wide range of other bacteria such as Rhodococcus and Streptomyces. In the majority of instances these are associated with CYP genes. These ferredoxin systems are important in controlling electron transfer across bacterial secondary metabolite production processes which include antibiotic and pigment formation among others.

Binding of a physiological substrate causes large-scale conformational reorganization in cytochrome P450 51.

Sterol 14α-demethylases (CYP51s) are phylogenetically the most conserved cytochromes P450, and their three-step reaction is crucial for biosynthesis of sterols and serves as a leading target for clinical and agricultural antifungal agents. The structures of several (bacterial, protozoan, fungal, and human) CYP51 orthologs, in both the ligand-free and inhibitor-bound forms, have been determined and have revealed striking similarity at the secondary and tertiary structural levels, despite having low sequence identity. Moreover, in contrast to many of the substrate-promiscuous, drug-metabolizing P450s, CYP51 structures do not display substantial rearrangements in their backbones upon binding of various inhibitory ligands, essentially representing a snapshot of the ligand-free sterol 14α-demethylase. Here, using the obtusifoliol-bound I105F variant of Trypanosoma cruzi CYP51, we report that formation of the catalytically competent complex with the physiological substrate triggers a large-scale conformational switch, dramatically reshaping the enzyme active site (3.5-6.0 Å movements in the FG arm, HI arm, and helix C) in the direction of catalysis. Notably, our X-ray structural analyses revealed that the substrate channel closes, the proton delivery route opens, and the topology and electrostatic potential of the proximal surface reorganize to favor interaction with the electron-donating flavoprotein partner, NADPH-cytochrome P450 reductase. Site-directed mutagenesis of the amino acid residues involved in these events revealed a key role of active-site salt bridges in contributing to the structural dynamics that accompanies CYP51 function. Comparative analysis of apo-CYP51 and its sterol-bound complex provided key conceptual insights into the molecular mechanisms of CYP51 catalysis, functional conservation, lineage-specific substrate complementarity, and druggability differences.

Structural and functional characterisation of the cytochrome P450 enzyme CYP268A2 from Mycobacterium marinum.

Members of the cytochrome P450 monooxygenase family CYP268 are found across a broad range of Mycobacterium species including the pathogens Mycobacterium avium, M. colombiense, M. kansasii, and Mmarinum CYP268A2, from M. marinum, which is the first member of this family to be studied, was purified and characterised. CYP268A2 was found to bind a variety of substrates with high affinity, including branched and straight chain fatty acids (C10-C12), acetate esters, and aromatic compounds. The enzyme was also found to bind phenylimidazole inhibitors but not larger azoles, such as ketoconazole. The monooxygenase activity of CYP268A2 was efficiently reconstituted using heterologous electron transfer partner proteins. CYP268A2 hydroxylated geranyl acetate and trans-pseudoionone at a terminal methyl group to yield (2E,6E)-8-hydroxy-3,7-dimethylocta-2,6-dien-1-yl acetate and (3E,5E,9E)-11-hydroxy-6,10-dimethylundeca-3,5,9-trien-2-one, respectively. The X-ray crystal structure of CYP268A2 was solved to a resolution of 2.0 Šwith trans-pseudoionone bound in the active site. The overall structure was similar to that of the related phytanic acid monooxygenase CYP124A1 enzyme from Mycobacterium tuberculosis, which shares 41% sequence identity. The active site is predominantly hydrophobic, but includes the Ser99 and Gln209 residues which form hydrogen bonds with the terminal carbonyl group of the pseudoionone. The structure provided an explanation on why CYP268A2 shows a preference for shorter substrates over the longer chain fatty acids which bind to CYP124A1 and the selective nature of the catalysed monooxygenase activity.