Crystal Structure of Albaflavenone Monooxygenase Containing a Moonlighting Terpene Synthase Active Site

Albaflavenone synthase (CYP170A1) is a monooxygenase catalyzing the final two steps in the biosynthesis of this antibiotic in the soil bacterium, Streptomyces coelicolor A3(2). Interestingly, CYP170A1 shows no stereo selection forming equal amounts of two albaflavenol epimers, each of which is oxidized in turn to albaflavenone. To explore the structural basis of the reaction mechanism, we have studied the crystal structures of both ligand-free CYP170A1 (2.6 Å) and complex of endogenous substrate (epi-isozizaene) with CYP170A1 (3.3 Å). The structure of the complex suggests that the proximal epi-isozizaene molecules may bind to the heme iron in two orientations. In addition, much to our surprise, we have found that albaflavenone synthase also has a second, completely distinct catalytic activity corresponding to the synthesis of farnesene isomers from farnesyl diphosphate. Within the cytochrome P450 α-helical domain both the primary sequence and x-ray structure indicate the presence of a novel terpene synthase active site that is moonlighting on the P450 structure. This includes signature sequences for divalent cation binding and an α-helical barrel. This barrel is unusual because it consists of only four helices rather than six found in all other terpene synthases. Mutagenesis establishes that this barrel is essential for the terpene synthase activity of CYP170A1 but not for the monooxygenase activity. This is the first bifunctional P450 discovered to have another active site moonlighting on it and the first time a terpene synthase active site is found moonlighting on another protein.     

Investigating conservation of the albaflavenone biosynthetic pathway and CYP170 bifunctionality in streptomycetes

Albaflavenone, a tricyclic sesquiterpene antibiotic, is biosynthesized in Streptomyces coelicolor A3(2) by enzymes encoded in a two-gene operon. Initially, sesquiterpene cyclase catalyzes the cyclization of farnesyl diphosphate to the terpenoid epi-isozizaene, which is oxidized to the final albaflavenone by cytochrome P450 (CYP)170A1. Additionally, this CYP is a bifunctional enzyme, being able to also generate farnesene isomers from farnesyl diphosphate, owing to a terpene synthase active site moonlighting on the CYP molecule. To explore the functionality of this operon in other streptomycetes, we have examined culture extracts by GC/MS and established the presence of albaflavenone in five Streptomyces species. Bioinformatics examination of the predicted CYP170 primary amino acid sequences revealed substitutions in the CYP terpene synthase active site. To examine whether the terpene synthase site was catalytically active in another CYP170, we characterized the least related CYP170 orthologue from Streptomyces albus (CYP170B1). Following expression and purification, CYP170B1 showed a normal reduced CO difference spectrum at 450 nm, in contrast to the unusual 440-nm peak observed for S. coelicolor A3(2) CYP170A1. CYP170B1 can catalyze the conversion of epi-isozizaene to albaflavenone, but was unable to catalyze the conversion of farnesyl diphosphate to farnesene. Molecular modeling with our crystal structure of CYP170A1 suggests that the absence of key amino acids for binding the essential terpene synthase cofactor Mg(2+) may be the explanation for the loss of CYP170B1 bifunctionality.     

A new method to purify hepatic CYP1A of Prochilodus scrofa, a Brazilian freshwater fish

Cytochromes P450 constitute a superfamily of the phase I enzymes whose primary task is the detoxification of both endogenous and xenobiotic compounds. Fish, among non-mammalian species, have received great interest because they are a direct food source for humans as well as conveyors of toxic chemicals to human beings. The aim of the present study was the purification of the hepatic isoform of CYP1A in Prochilodus scrofa (Prochilodontidae), a Brazilian fish, using only one chromatographic step. The purification of CYP1A was done by Reverse Phase HPLC on a C18 column. Purified CYP1A was characterized with respect to electrophoretic, immunochemical and biocatalyst properties. CYP1A fractions produced a single uniform band on SDS-PAGE with an apparent molecular mass of 58 kDa. Purified CYP1A of P. scrofa showed strong cross-reactivity with antibodies directed against CYP1A from trout. The fraction was also encapsulated in two different reconstituted systems; one composed of neutral lipids and another of negatively charged lipids. In both of them, we could detect EROD activity but not PROD activity, which confirms that the CYP1A was purified with all its enzyme activity. There was an increase of activity when CYP1A and NADPH cytochrome P450 (CYP) reductase were encapsulated in negatively charged lipids, which confirms that the charge of lipid is essential to CYP1A activity. All these characteristics strongly suggest that this new procedure is efficient for purifying hepatic CYP1A from P. scrofa, showing that the CYP1A isoform of this fish has a highly conserved protein region.     

Substrate specificity of the cytochrome P450 enzymes CYP79A1 and CYP71E1 involved in the biosynthesis of the cyanogenic glucoside dhurrin in Sorghum bicolor (L.) Moench

The two multifunctional cytochrome P450 enzymes, CYP79A1 and CYP71E1, involved in the biosynthesis of the cyanogenic glucoside dhurrin in Sorghum bicolor (L.) Moench have been characterized with respect to substrate specificity and cofactor requirements using reconstituted, recombinant enzymes and sorghum microsomes. CYP79A1 has a very high substrate specificity, tyrosine being the only substrate found. CYP71E1 has less stringent substrate requirements and metabolizes aromatic oximes efficiently, whereas aliphatic oximes are slowly metabolized. Neither CYP79A1 nor CYP71E1 catalyze the metabolism of a range of different herbicides. The reported resistance of sorghum to bentazon is therefore not linked to the presence of CYP79A1 or CYP71E1. NADPH is a much better cofactor than NADH although NADH does support the entire catalytic cycle of both P450 enzymes. Km and Vmax values for NADPH when supporting CYP71E1 activity are 0.013 mM and 111 nmol/mg protein/s. For NADH, the corresponding values are 0. 3 mM and 42 nmol/mg protein/s. CYP79A1 is a fairly stable enzyme. In contrast, CYP71E1 is labile and prone to rapid denaturation at room temperature. CYP71E1 is isolated in the low spin form. CYP71E1 catalyzes an unusual dehydration reaction of an oxime to the corresponding nitrile which subsequently is C-hydroxylated. The oxime forms a peculiar reverse Type I spectrum, whereas the nitrile forms a Type I spectrum. Several compounds which do not serve as substrates formed Type I substrate binding spectra with the two P450 enzymes.     

A gel-free MS-based quantitative proteomic approach accurately measures cytochrome P450 protein concentrations in human liver microsomes

The human cytochrome P450 (P450) superfamily consists of membrane-bound proteins that metabolize a myriad of xenobiotics and endogenous compounds. Quantification of P450 expression in various tissues under normal and induced conditions has an important role in drug safety and efficacy. Conventional immunoquantification methods have poor dynamic range, low throughput, and a limited number of specific antibodies. Recent advances in MS-based quantitative proteomics enable absolute protein quantification in a complex biological mixture. We have developed a gel-free MS-based protein quantification strategy to quantify CYP3A enzymes in human liver microsomes (HLM). Recombinant protein-derived proteotypic peptides and synthetic stable isotope-labeled proteotypic peptides were used as calibration standards and internal standards, respectively. The lower limit of quantification was approximately 20 fmol P450. In two separate panels of HLM examined (n = 11 and n = 22), CYP3A, CYP3A4 and CYP3A5 concentrations were determined reproducibly (CV or=0.87) and marker activities (r(2)>or=0.88), including testosterone 6beta-hydroxylation (CYP3A), midazolam 1'-hydroxylation (CYP3A), itraconazole 6-hydroxylation (CYP3A4) and CYP3A5-mediated vincristine M1 formation (CYP3A5). Taken together, our MS-based method provides a specific, sensitive and reliable means of P450 protein quantification and should facilitate P450 characterization during drug development, especially when specific substrates and/or antibodies are unavailable.     

CYP11A1 stimulates the hydroxylase activity of CYP11B1 in mitochondria of recombinant yeast in vivo and in vitro.

In mammals, hydrocortisone synthesis from cholesterol is catalyzed by a set of five specialized enzymes, four of them belonging to the superfamily of cytochrome P-450 monooxygenases. A recombinant yeast expression system was recently developed for the CYP11B1 (P45011beta) enzyme, which performs the 11beta hydroxylation of steroids such as 11-deoxycortisol into hydrocortisone, one of the three mitochondrial cytochrome P-450 proteins involved in steroidogenesis in mammals. This heterologous system was used to test the potential interaction between CYP11B1 and CYP11A1 (P450scc), the mitochondrial cytochrome P-450 enzyme responsible for the side chain cleaving of cholesterol. Recombinant CYP11B1 and CYP11A1 were targeted to Saccharomyces cerevisiae mitochondria using the yeast cytochrome oxidase subunit 6 mitochondrial presequence fused to the mature form of the two proteins. In yeast, the presence of CYP11A1 appears to improve 11beta hydroxylase activity of CYP11B1 in vivo and in vitro. Fractionation experiments indicate the presence of the two proteins in the same membrane fractions, i.e. inner membrane and contact sites of mitochondria. Thus, yeast mitochondria provide interesting insights to study some molecular and cellular aspects of mammalian steroid synthesis. In particular, recombinant yeast should permit a better understanding of the mechanism permitting the synthesis of steroids (sex steroids, mineralocorticoids and glucocorticoids) with a minimal set of enzymes at physiological level, thus avoiding disease states.     

Atomic force microscopy visualization and measurement of the activity and physicochemical properties of single monomeric and oligomeric enzymes

An approach to measure the activity of single oligomers of the heme-containing enzyme cytochrome P450 CYP102A1 (CYP102A1) by atomic force microscopy (AFM) has been developed. It was found that the amplitude of fluctuations of the height of single CYP102A1 molecules performing the catalytic cycle is twice as great as the amplitude of fluctuations of the height of the same enzymes in the inactive state. It was shown that the amplitude of height fluctuations of a CYP102A1 protein globule depends on temperature, the maximum of this dependence being observed at 22°C. The activity of a single CYP102A1 molecule in the unit amplitude of height fluctuations of a protein globule per unit time was 5 ± 2 protein molecule was measured from the deformation of this molecule by the action of an AFM probe. The use of AFM probes of different geometry made it possible to determine the integral and local Young’s modulus for the monomers of the protein putidaredoxin reductase from the cytochrome P450 CYP101 (P450cam)-containing monooxigenase system, which were 37 ± 117 and 1 ± 3 MPa, respectively.     

Assessment of P450 induction in the marmoset monkey using targeted anti-peptide antibodies

The identity and expression of hepatic P450 enzymes in marmosets was investigated using a panel of anti-peptide antibodies originally targeted against human P450 enzymes. In immunoblotting, of 12 antibodies examined, 10 bound specifically to bands in marmoset liver microsomal fraction corresponding to P450 enzymes. It is proposed that these represent marmoset CYP1A1, CYP1A2, CYP2A, CYP2B, CYP2C forms (CYP2C-1 and CYP2C-2), CYP2D19, CYP3A21 and another CYP3A form (CYP3A-m). The antibodies, together with an anti-marmoset CYP2E1 antibody, were used to investigate the expression of 10 P450 enzymes in marmosets treated with P450-inducing chemicals. Treatment with phenobarbitone caused CYP2B, CYP2C-2 and CYP3A21 levels to increase, rifampicin caused increases in CYP2B and CYP2C-1 and a decrease in CYP3A21 levels, whereas dioxin caused CYP1A1 and CYP1A2 levels to increase and CYP2E1 levels to decrease. Clofibric acid did not induce any P450. P450 enzyme activities were assessed using 8 different substrates and increases were found after treatment with phenobarbitone, rifampicin, and dioxin. However, due to species differences in substrate selectivity, it proved difficult to ascribe these changes to individual P450 enzymes. Thus, the use of anti-peptide antibodies provides a more informative way of assessing the levels of specific P450 enzymes than enzyme activity measurements.     

Effect of homomeric P450-P450 complexes on P450 function

Previous studies have shown that the presence of one P450 enzyme can affect the function of another. The goal of the present study was to determine if P450 enzymes are capable of forming homomeric complexes that affect P450 function. To address this problem, the catalytic activities of several P450s were examined in reconstituted systems containing NADPH-POR (cytochrome P450 reductase) and a single P450. CYP2B4 (cytochrome P450 2B4)-, CYP2E1 (cytochrome P450 2E1)- and CYP1A2 (cytochrome P450 1A2)-mediated activities were measured as a function of POR concentration using reconstituted systems containing different concentrations of P450. Although CYP2B4-dependent activities could be explained by a simple Michaelis-Menten interaction between POR and CYP2B4, both CYP2E1 and CYP1A2 activities generally produced a sigmoidal response as a function of [POR]. Interestingly, the non-Michaelis behaviour of CYP1A2 could be converted into a simple mass-action response by increasing the ionic strength of the buffer. Next, physical interactions between CYP1A2 enzymes were demonstrated in reconstituted systems by chemical cross-linking and in cellular systems by BRET (bioluminescence resonance energy transfer). Cross-linking data were consistent with the kinetic responses in that both were similarly modulated by increasing the ionic strength of the surrounding solution. Taken together, these results show that CYP1A2 forms CYP1A2-CYP1A2 complexes that exhibit altered catalytic activity.     

Quantification of cholesterol-metabolizing P450s CYP27A1 and CYP46A1 in neural tissues reveals a lack of enzyme-product correlations in human retina but not human brain

Cytochrome P450 enzymes (CYP or P450) 46A1 and 27A1 play important roles in cholesterol elimination from the brain and retina, respectively, yet they have not been quantified in human organs because of their low abundance and association with membrane. On the basis of our previous development of a multiple reaction monitoring (MRM) workflow for measurements of low-abundance membrane proteins, we quantified CYP46A1 and CYP27A1 in human brain and retina samples from four donors. These enzymes were quantified in the total membrane pellet, a fraction of the whole tissue homogenate, using 1?N-labled recombinant P450s as internal standards. The average P450 concentrations/mg of total tissue protein were 345 fmol of CYP46A1 and 110 fmol of CYP27A1 in the temporal lobe, and 60 fmol of CYP46A1 and 490 fmol of CYP27A1 in the retina. The corresponding P450 metabolites were then measured in the same tissue samples and compared to the P450 enzyme concentrations. Investigation of the enzyme-product relationships and analysis of the P450 measurements based on different signature peptides revealed a possibility of retina-specific post-translational modification of CYP27A1. The data obtained provide important insights into the mechanisms of cholesterol elimination from different neural tissues.     

Molecular cloning and nucleotide sequence of a new P450 gene, CYP319A1, from the cattle tick, Boophilus microplus.

We have isolated and sequenced a novel P450 gene (CYP319A1) from the cattle tick, Boophilus microplus. The CYP319A1 cDNA encodes a protein of 531 amino acids with an estimated molecular weight of 60.9k. It contains all highly conserved motifs characteristic of P450 enzymes. Comparison of deduced amino acid sequence with other CYP members shows that the CYP319A1 is more closely related to CYP4 family, but its overall identity to the CYP4 family is less than 40%. Therefore, it was assigned to a new P450 family by the P450 nomenclature committee. A pseudogene which shares high homology with the CYP319A1 was identified. Analysis of genomic sequence of the pseudogene indicated that the pseudogene contains two additional DNA inserts in the coding region, which disrupt the open reading frame. RT-PCR analysis showed that CYP319A1 is expressed in both susceptible and acaricide-resistant ticks.     

P450 enzymes from the bacterium Novosphingobium aromaticivorans

Twelve of the fifteen potential P450 enzymes from the bacterium Novosphingobium aromaticivorans, which is known to degrade a wide range of aromatic hydrocarbons, have been produced via heterologous expression in Escherichia coli. The enzymes were tested for their ability to bind a range of substrates including polyaromatic hydrocarbons. While two of the enzymes were found to bind aromatic compounds (CYP108D1 and CYP203A2), the others show binding with a variety of compounds including linear alkanes (CYP153C1) and mono- and sesqui-terpenoid compounds (CYP101B1, CYP101C1, CYP101D1, CYP101D2, CYP111A1, and CYP219A1). A 2Fe-2S ferredoxin (Arx-A), which is associated with CYP101D2, was also produced. The activity of five of the P450 enzymes (CYP101B1, CYP101C1, CYP101D1, CYP101D2, and CYP111A2) was reconstituted with Arx-A and putidaredoxin reductase (of the P450cam system from Pseudomonas putida) in a Class I type electron transfer system. Preliminary characterisation of the majority of the substrate oxidation products is reported.     

Deletion of P399_E401 in NADPH cytochrome P450 oxidoreductase results in partial mixed oxidase deficiency

P450 oxidoreductase (POR) is the electron donor for all microsomal P450s including steroidogenic enzymes CYP17A1, CYP19A1 and CYP21A2. We found a novel POR mutation P399_E401del in two unrelated Turkish patients with 46,XX disorder of sexual development. Recombinant POR proteins were produced in yeast and tested for their ability to support steroid metabolizing P450 activities. In comparison to wild-type POR, the P399_E401del protein was found to decrease catalytic efficiency of 21-hydroxylation of progesterone by 68%, 17α-hydroxylation of progesterone by 76%, 17,20-lyase action on 17OH-pregnenolone by 69%, aromatization of androstenedione by 85% and cytochrome c reduction activity by 80%. Protein structure analysis of the three amino acid deletion P399_E401 revealed reduced stability and flexibility of the mutant. In conclusion, P399_E401del is a novel mutation in POR that provides valuable genotype–phenotype and structure–function correlation for mutations in a different region of POR compared to previous studies. Characterization of P399_E401del provides further insight into specificity of different P450s for interaction with POR as well as nature of metabolic disruptions caused by more pronounced effect on specific P450s like CYP17A1 and aromatase.     

Induction of cytochrome P450 1A1 and conjugating enzymes in rainbow trout (Oncorhynchus mykiss) liver: A time course study

1. The effects of i.p. injections of isosafrole (ISF) or β-naphthoflavone (β-NF) on the cytochrome P450 (CYP) 1A1 system and conjugating enzymes were investigated in livers from juvenile rainbow trout in a time course study employing catalytic, immunochemical and cDNA probes.2. β-NF treatment resulted in a rapid rise in CYP1A1 mRNA followed by accumulation of P450 1A1 protein and P450 1A1 mediated enzyme activity measured as ethoxyresorufin-O-deethylase (EROD) activity.3. ISF treatment resulted in a comparatively weak induction of CYP1A1 mRNA and P450 1A1 protein levels whilst EROD activity was markedly induced; thus when expressed on the basis of immunoquantified P450 1A1 protein, the specific EROD activity was signficantly higher in ISF than β-NF treated fish.4. In vitro inhibition studies revealed that ISF inhibited EROD activity to a far lesser extent than β-NF.5. Conjugation enzymes represented by phenol UDP-glucuronosyltransferase and glutathione S-transferase (GST) activities, were induced by β-NF, whereas ISF treatment had no effect on these enzyme activities.6. Immunoblotting using antibodies raised against rat GST7-7 showed that a Pi class trout GST enzyme was induced by β-NF treatment.     

The CYP701B1 of Physcomitrella patens is an ent-kaurene oxidase that resists inhibition by uniconazole-P

The moss Physcomitrella patens produces both ent-kaurene and ent-kaurenoic acid, which are intermediates of gibberellin biosynthesis in flowering plants. The CYP701 superfamily of cytochrome P450s functions as ent-kaurene oxidases in the biosynthesis of ent-kaurenoic acid. A candidate gene encoding ent-kaurene oxidase in P. patens, CYP701B1, was cloned and heterologously expressed in yeast to examine enzyme activities in vitro. The recombinant CYP701B1 protein catalyzed the oxidation reaction from ent-kaurene to ent-kaurenoic acid. CYP701B1 activity was highly resistant to the ent-kaurene oxidase inhibitor uniconazole-P (IC(50) 64 μM), even though the activity of Arabidopsis ent-kaurene oxidase (CYP701A3) was sensitive (IC(50) 0.26 μM).     

A novel sterol 14alpha-demethylase/ferredoxin fusion protein (MCCYP51FX) from Methylococcus capsulatus represents a new class of the cytochrome P450 superfamily

Sterol 14alpha-demethylase encoded by CYP51 is a member of the cytochrome P450 (CYP) superfamily of enzymes and has been shown to have an essential role in sterol biosynthesis in eukaryotes, with orthologues recently being described in some bacteria. Examination of the genome sequence data for the proteobacterium Methylococcus capsulatus, a bacterial species known to produce sterol, revealed the presence of a single CYP with strong homology to CYP51, particularly to a form in Mycobacterium tuberculosis. This M. capsulatus CYP51 protein represents a new class of CYP consisting of the CYP domain naturally fused to a ferredoxin domain at the C terminus via an alanine-rich linker. Expression of the M. capsulatus MCCYP51FX fusion in Escherichia coli yielded a P450, which, when purified to homogeneity, had the predicted molecular mass approximately 62 kDa on SDS/PAGE and bound lanosterol as a putative substrate. Sterol 14alpha-demethylase activity was shown (0.24 nmol of lanosterol metabolized per minute per nanomole of MCCYP51FX fusion) by gas chromatography/mass spectrometry with the activity dependent upon the presence of ferredoxin reductase and NADPH. Our unique findings describe a new class of naturally existing cytochrome P450, which will provide pivotal information for CYP structure/function in general.