Microsomal Cytochrome P450 Monooxygenase Research Articles

The Disconnect in Intrinsic Clearance Determined in Human Hepatocytes and Liver Microsomes Results from Divergent Cytochrome P450 Activities.

Candidate drugs may exhibit higher unbound intrinsic clearances (CLint,u) in human liver microsomes (HLMs) relative to human hepatocytes (HHs), posing a challenge as to which value is more predictive of in vivo clearance (CL). This work was aimed at better understanding the mechanism(s) underlying this 'HLM:HH disconnect' via examination of previous explanations, including passive permeability limited CL or cofactor exhaustion in hepatocytes. A series of structurally related, passively permeable (Papps > 5 × 10-6 cm/s), 5-azaquinazolines were studied in different liver fractions, and metabolic rates and routes were determined. A subset of these compounds demonstrated a significant HLM:HH (CLint,u ratio 2-26) disconnect. Compounds were metabolized via combinations of liver cytosol aldehyde oxidase (AO), microsomal cytochrome P450 (CYP) and flavin monooxygenase (FMO). For this series, the lack of concordance between CLint,u determined in HLM and HH contrasted with an excellent correlation of AO dependent CLint,u determined in human liver cytosol[Formula: see text], r2 = 0.95, P < 0.0001). The HLM:HH disconnect for both 5-azaquinazolines and midazolam was as a result of significantly higher CYP activity in HLM and lysed HH fortified with exogenous NADPH relative to intact HH. Moreover, for the 5-azaquinazolines, the maintenance of cytosolic AO and NADPH-dependent FMO activity in HH, relative to CYP, supports the conclusion that neither substrate permeability nor intracellular NADPH for hepatocytes were limiting CLint,u Further studies are required to identify the underlying cause of the lower CYP activities in HH relative to HLM and lysed hepatocytes in the presence of exogenous NADPH. SIGNIFICANCE STATEMENT: Candidate drugs may exhibit higher intrinsic clearance in human liver microsomes relative to human hepatocytes, posing a challenge as to which value is predictive of in vivo clearance. This work demonstrates that the difference in activity determined in liver fractions results from divergent cytochrome P450 but not aldehyde oxidase or flavin monooxygenase activity. This is inconsistent with explanations including substrate permeability limitations or cofactor exhaustion and should inform the focus of further studies to understand this cytochrome P450 specific disconnect phenomenon.

Towards understanding transfluthrin efficacy in a pyrethroid-resistant strain of the malaria vector Anopheles funestus with special reference to cytochrome P450-mediated detoxification

Malaria vector control interventions rely heavily on the application of insecticides against anopheline mosquitoes, in particular the fast-acting pyrethroids that target insect voltage-gated sodium channels (VGSC). Frequent applications of pyrethroids have resulted in resistance development in the major malaria vectors including Anopheles funestus, where resistance is primarily metabolic and driven by the overexpression of microsomal cytochrome P450 monooxygenases (P450s). Here we examined the pattern of cross-resistance of the pyrethroid-resistant An. funestus strain FUMOZ-R towards transfluthrin and multi-halogenated benzyl derivatives, permethrin, cypermethrin and deltamethrin in comparison to the susceptible reference strain FANG. Transfluthrin and two multi-fluorinated derivatives exhibited micromolar potency - comparable to permethrin - to functionally expressed dipteran VGSC in a cell-based cation influx assay. The activity of transfluthrin and its derivatives on VGSC was strongly correlated with their contact efficacy against strain FUMOZ-R, although no such correlation was obtained for the other pyrethroids due to their rapid detoxification by the resistant strain. The low resistance levels for transfluthrin and derivatives in strain FUMOZ-R were only weakly synergized by known P450 inhibitors such as piperonyl butoxide (PBO), triflumizole and 1-aminobenzotriazole (1-ABT). In contrast, deltamethrin toxicity in FUMOZ-R was synergized > 100-fold by all three P450 inhibitors. The biochemical profiling of a range of fluorescent resorufin and coumarin compounds against FANG and FUMOZ-R microsomes identified 7-benzyloxymethoxy-4-trifluoromethylcoumarin (BOMFC) as a highly sensitive probe substrate for P450 activity. BOMFC was used to develop a fluorescence-based high-throughput screening assay to measure the P450 inhibitory action of potential synergists. Azole fungicides prochloraz and triflumizole were identified as extremely potent nanomolar inhibitors of microsomal P450s, strongly synergizing deltamethrin toxicity in An. funestus. Overall, the present study contributed to the understanding of transfluthrin efficacy at the molecular and organismal level and identified azole compounds with potential to synergize pyrethroid efficacy in malaria vectors.

Biosensor selection of small compounds, modulating a complex formation between cytochrome P450s and NADPH-dependent P450 oxidoreductase

The study of the effect of low-molecular-weight compounds (substrates, endogenous metabolites, drugs and xenobiotics) on the kinetic and equilibrium parameters of functionally significant binary protein-protein interactions (PPIs) is of both fundamental and clinical importance. The surface plasmon resonance (SPR) is the method of the first choice for studying PPIs. Earlier, SPR analysis revealed the modulating effect of steroidal substrates on the affinity of interactions between steroidogenic microsomal cytochromes P450 (CYP) and their redox partner (cytochrome b5). In this work, we have shown the suitability of the experimental approach for assessing the selective effect of the cofactor NADPH on the interaction between cytochromes CYP3A4 or CYP2E1 with NADPH-dependent P450 oxidoreductase (CPR). Experiments have shown that the CYP3A4/CPR complex is not modulated by NADPH, while the dissociation rate of the CYP2E1/CPR complex in the presence of NADPH significantly decreased: the koff values in the absence and presence of NADPH were (3.6 ± 0.2) • 10-3 s-1 and (3.8 ± 0.2) • 10-4 s-1, respectively. Thus, in the presence of NADPH, an increase in the affinity of CYP2E1/CPR complex formation by approximately one order of magnitude was observed, while NADPH did not affect the kon value of this complex. Co-injection of NADPH at the CYP2E1/CPR complex preformed in the absence of NADPH had minor influence on the koff values (&lt;10%). This suggests a stabilizing role of NADPH for the CYP2E1/CPR complex formation. Thus, the use of our approach made it possible to assess the effect of the main electron supplier for the microsomal cytochrome P450 monooxygenase system on the kinetic rate constants of CYP/CPR complexes.

Structural and Functional Studies of the Membrane-Binding Domain of NADPH-Cytochrome P450 Oxidoreductase.

NADPH-cytochrome P450 oxidoreductase (CYPOR), the essential flavoprotein of the microsomal cytochrome P450 monooxygenase system, is anchored in the phospholipid bilayer by its amino-terminal membrane-binding domain (MBD), which is necessary for efficient electron transfer to cytochrome P450. Although crystallographic and kinetic studies have established the structure of the soluble catalytic domain and the role of conformational motions in the control of electron transfer, the role of the MBD is largely unknown. We examined the role of the MBD in P450 catalysis through studies of amino-terminal deletion mutants and site-directed spin labeling. We show that the MBD spans the membrane and present a model for the orientation of CYPOR on the membrane capable of forming a complex with cytochrome P450. EPR power saturation measurements of CYPOR mutants in liposomes containing a lipid/Ni(II) chelate identified a region of the soluble domain interacting with the membrane. The deletion of more than 29 residues from the N-terminus of CYPOR decreases cytochrome P450 activity concomitant with alterations in electrophoretic mobility and an increased resistance to protease digestion. The altered kinetic properties of these mutants are consistent with electron transfer through random collisions rather than via formation of a stable CYPOR-P450 complex. Purified MBD binds weakly to cytochrome P450, suggesting that other interactions are also required for CYPOR-P450 complex formation. We propose that the MBD and flexible tether region of CYPOR, residues 51-63, play an important role in facilitating the movement of the soluble domain relative to the membrane and in promoting multiple orientations that permit specific interactions of CYPOR with its varied partners.

Phototrophic production of heterologous diterpenoids and a hydroxy-functionalized derivative from Chlamydomonas reinhardtii

Photosynthetic microalgae harbor enormous potential as light-driven green-cell factories for sustainable bio-production of a range of natural and heterologous products such as isoprenoids. Their capacity for photosynthesis and rapid low-input growth with (sun)light and CO2 is coupled to a robust metabolic architecture structured toward the generation of isoprenoid pigments and compounds involved in light capture, electron transfer, and radical scavenging. Metabolic engineering approaches using eukaryotic green microalgae have previously been hampered mainly by low-levels of nuclear transgene expression. Here, we employed a strategy of optimized transgene design which couples codon optimization and synthetic intron spreading for the expression of heterologous plant enzymes from the algal nuclear genome. The diterpenoids casbene, taxadiene, and 13R(+) manoyl oxide were produced after expressing heterologous diterpene synthases and enzymes participating in the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway which were all targeted to the algal chloroplast. Additionally, a truncated and soluble plant microsomal cytochrome P450 monooxygenase was functionally expressed and able to hydroxylate 13R(+) manoyl oxide when directed into the chloroplasts. The heterologous diterpenoids were found to be excreted from the cells and accumulate in dodecane solvent-culture overlays. It was shown that the algal cell could tolerate significant metabolic pull towards diterpenoids without loss of native pigments. Using an algal strain producing 13R(+) manoyl oxide as a model, diterpenoid production was shown to be highest in photoautotrophic cultivations using CO2 as the sole carbon source and day:night illumination cycles. Up to 80 mg 13R(+) manoyl oxide per gram cell dry mass (CDM) could be produced from C. reinhardtii in a 7 day batch cultivation with a sustained maximal productivity of 22.5 mg gcdm−1 d−1 over 3 consecutive days. Collectively the results presented here suggest that green algal cells have remarkable potential for the heterologous production of non-native isoprenoids and support the use of these hosts for (sun)light driven bioproduction concepts.

Membrane Associated Progesterone Receptors: Promiscuous Proteins with Pleiotropic Functions - Focus on Interactions with Cytochromes P450.

Membrane-associated progesterone receptors (MAPR) are a group of four rather small, partially homologous proteins, which share a similar non-covalent heme-binding domain that is related to cytochrome b5, a well-known functional interaction partner of microsomal cytochrome P450 (CYP) monooxygenase systems. Apart from their structural similarities the four proteins progesterone membrane component 1 (PGRMC1, also referred to as IZA, sigma-2 receptor, Dap1), PGRMC2, neudesin (NENF) and neuferricin (CYB5D2) display surprisingly divergent and multifunctional physiological properties related to cholesterol/steroid biosynthesis, drug metabolism and response, iron homeostasis, heme trafficking, energy metabolism, autophagy, apoptosis, cell cycle regulation, cell migration, neural functions, and tumorigenesis and cancer progression. The purpose of this mini-review is to briefly summarize the structural and functional properties of MAPRs with particular focus on their interactions with the CYP system. For PGRMC1, originally identified as a non-canonical progesterone-binding protein that mediates some immediate non-genomic actions of progesterone, available evidence indicates mainly activating interactions with steroidogenic CYPs including CYP11A1, CYP21A2, CYP17, CYP19, CYP51A1, and CYP61A1, while interactions with drug metabolizing CYPs including CYP2C2, CYP2C8, CYP2C9, CYP2E1, and CYP3A4 were either ineffective or slightly inhibitory. For the other MAPRs the evidence is so far less conclusive. We also point out that experimental limitations question some of the previous conclusions. Use of appropriate model systems should help to further clarify the true impact of these proteins on CYP-mediated metabolic pathways.

Decreased bile-acid synthesis in livers of hepatocyte-conditional NADPH-cytochrome P450 reductase-null mice results in increased bile acids in serum.

NADPH-cytochrome P450 reductase (Cpr) is essential for the function of microsomal cytochrome P450 monooxygenases (P450), including those P450s involved in bile acid (BA) synthesis. Mice with hepatocyte-specific deletion of NADPH-cytochrome P450 reductase (H-Cpr-null) have been engineered to understand the in vivo function of hepatic P450s in the metabolism of xenobiotics and endogenous compounds. However, the impact of hepatic Cpr on BA homeostasis is not clear. The present study revealed that H-Cpr-null mice had a 60% decrease in total BA concentration in liver, whereas the total BA concentration in serum was almost doubled. The decreased level of cholic acid (CA) in both serum and livers of H-Cpr-null mice is likely due to diminished enzyme activity of Cyp8b1 that is essential for CA biosynthesis. Feedback mechanisms responsible for the reduced liver BA concentrations and/or increased serum BA concentrations in H-Cpr-null mice included the following: 1) enhanced alternative BA synthesis pathway, as evidenced by the fact that classic BA synthesis is diminished but chenodeoxycholic acid still increases in both serum and livers of H-Cpr-null mice; 2) inhibition of farnesoid X receptor activation, which increased the mRNA of Cyp7a1 and 8b1; 3) induction of intestinal BA transporters to facilitate BA absorption from the intestine to the circulation; 4) induction of hepatic multidrug resistance-associated protein transporters to increase BA efflux from the liver to blood; and 5) increased generation of secondary BAs. In summary, the present study reveals an important contribution of the alternative BA synthesis pathway and BA transporters in regulating BA concentrations in H-Cpr-null mice.

RNA interference of NADPH-cytochrome P450 reductase of the rice brown planthopper,Nilaparvata lugens,increases susceptibility to insecticides

NADPH-cytochrome P450 reductase (CPR) is essential for numerous biological reactions catalysed by microsomal cytochrome P450 monooxygenases (P450s). Knockdown of CPR in several insects leads to developmental defects and increased susceptibility to insecticides. However, information about the role of CPR in the brown planthopper, Nilaparvata lugens, is still unavailable. A full-length cDNA encoding CPR was cloned from N. lugens (NlCPR). The deduced amino acid sequence showed marked features of classical CPRs, such as an N-terminus membrane anchor, conserved domains for flavin mononucleotide, flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide phosphate binding, as well as an FAD-binding motif and catalytic residues. Phylogenetic analysis revealed that NlCPR was located in a branch along with bed bug and pea aphid hemipteran insects. NlCPR mRNA was detectable in all tissues and developmental stages of N. lugens, as determined by real-time quantitative PCR. NlCPR transcripts were most abundant in the abdomen in adults, and in first-instar nymphs. Injection of N. lugens with double-strand RNA (dsRNA) against NlCPR significantly reduced the transcription level of the mRNA, and silencing of NlCPR resulted in increased susceptibility in N. lugens to beta-cypermethrin and imidacloprid. The results provide first evidence that NlCPR contributes to the susceptibility to beta-cypermethrin and imidacloprid in N. lugens.

Reponses of the Hepatic Microsomal Cytochrome P450 Monooxygenase System in Rock Bream Oplegnathus fasciatus Exposed to Tributyltin (TBT)

Abstract The study was conducted to investigate the responses of the hepatic microsomal cytochrome P450 monooxygenase system in the rock bream Oplegnathus fasciatus after chronic exposure to 0, 1, 2, 4, and 8 μg/L tributyltin (TBT) concentrations for 4 weeks. Hepatic cytochrome 450 content and ethoxyresorufin O -deethylation (EROD) activity were found to significantly increase in fish treated with the higher concentration of TBT (≥4 μg/L); however, no significant changes were observed in penthoxyresorufin O -deethylation (PROD) activity in all treated groups compared to the control group. These findings suggest that exposure to a low TBT concentration (≥4 μg/L) has the potential to induce cytochrome 450 content and EROD enzyme activity in hepatic tissue in the rock bream. Key words: Oplegnathus fasciatus , Tributyltin, Cytochrome 450, EROD, PROD Introduction Many aquatic ecosystems are faced with spatially or tem-porally alarmingly high levels of xenobiotic chemicals (Brack et al., 2002; Diez et al., 2002). Tributyltin (TBT) is a typi-cal synthetic pollutant, which was selected for investigation in this study. TBT is an organotin compound used primarily as a biocide in antifouling paints for ships, boats, and fishing nets. The use of TBT as an antifouling agent in paint has been banned for small boats and fishing nets in most countries as it has been shown to be toxic to aquatic life and is an endo-crine disrupting chemical that generates severe reproductive effects in aquatic animals (Horiguchi et al., 1994). Reproduc-tive impairment in gastropods has been reported at low TBT concentrations (Gibbs et al., 1988). TBT has been shown to induce imposex and intersex in brosobranchs (Oehlmann et al., 1998), and impaired reproductive function in fish has also been described (Mc Allister and Kime, 2003).Some studies have reported toxic effects of TBT such as morphological and functional alterations of teleost gills in aquatic media (Byrne et al., 1989; Schwaiger et al., 1992; Tsu -da et al., 1992; Wang and Huang, 1998). TBT has also been shown to markedly inhibit fish hepatic CYP and in particu-lar the isozyme cytochrome P4501A1 (CYP1A), both in vivo and vitro (Fent and Bucheli, 1994; Morcillo and Porte, 1997). The CYP1A isozyme plays a key role in the biotransformation of xenobiotic compounds, leading to either their detoxifica-tion or bioactivation, while other CYP forms are important

Biosynthesis of the biphenyl phytoalexin aucuparin in Sorbus aucuparia cell cultures treated with Venturia inaequalis

Aucuparin is the most widely distributed biphenyl phytoalexin in the rosaceous subtribe Pyrinae, which includes the economically important fruit trees apple and pear. The biphenyl scaffold is formed by biphenyl synthase, which catalyzes biosynthesis of 3,5-dihydroxybiphenyl. Conversion of this precursor to aucuparin (3,5-dimethoxy-4-hydroxybiphenyl) was studied in cell cultures of Sorbus aucuparia after treatment with an elicitor preparation from the scab-causing fungus Venturia inaequalis. The sequence of the biosynthetic steps detected was O-methylation - 4-hydroxylation - O-methylation. The two alkylation reactions were catalyzed by distinct methyltransferases, which differed in pH and temperature optima as well as stability. Biphenyl 4-hydroxylase was a microsomal cytochrome P450 monooxygenase, whose activity was appreciably decreased by the addition of established P450 inhibitors. When fed to V. inaequalis-treated S. aucuparia cell cultures, radioactively labeled 3,5-dihydroxybiphenyl was not only incorporated into aucuparin but also into the dibenzofuran eriobofuran, the accumulation of which paralleled that of aucuparin. However, biphenyl 2'-hydroxylase activity proposed to be involved in dibenzofuran formation was detected in neither microsomes nor cell-free extracts in the presence of NADPH and 2-oxoglutarate, respectively. Nevertheless, a basis for studying biphenyl biosynthesis at the gene level is provided.

Cross-Linking Mass Spectrometry and Mutagenesis Confirm the Functional Importance of Surface Interactions between CYP3A4 and Holo/Apo Cytochrome b5

Cytochrome b(5) (cyt b(5)) is one of the key components in the microsomal cytochrome P450 monooxygenase system. Consensus has not been reached about the underlying mechanism of cyt b(5) modulation of CYP catalysis. Both cyt b(5) and apo b(5) are reported to stimulate the activity of several P450 isoforms. In this study, the surface interactions of both holo and apo b(5) with CYP3A4 were investigated and compared for the first time. Chemical cross-linking coupled with mass spectrometric analysis was used to identify the potential electrostatic interactions between the protein surfaces. Subsequently, the models of interaction of holo/apo b(5) with CYP3A4 were built using the identified interacting sites as constraints. Both cyt b(5) and apo b(5) were predicted to bind to the same groove on CYP3A4 with close contacts to the B-B' loop of CYP3A4, a substrate recognition site. Mutagenesis studies further confirmed that the interacting sites on CYP3A4 (Lys96, Lys127, and Lys421) are functionally important. Mutation of these residues reduced or abolished cyt b(5) binding affinity. The critical role of Arg446 on CYP3A4 in binding to cyt b(5) and/or cytochrome P450 reductase was also discovered. The results indicated that electrostatic interactions on the interface of the two proteins are functionally important. The results indicate that apo b(5) can dock with CYP3A4 in a manner analogous to that of holo b(5), so electron transfer from cyt b(5) is not required for its effects.

Potential Biological Functions of Cytochrome P450 Reductase-dependent Enzymes in Small Intestine: NOVEL LINK TO EXPRESSION OF MAJOR HISTOCOMPATIBILITY COMPLEX CLASS II GENES

NADPH-cytochrome P450 reductase (POR) is essential for the functioning of microsomal cytochrome P450 (P450) monooxygenases and heme oxygenases. The biological roles of the POR-dependent enzymes in the intestine have not been defined, despite the wealth of knowledge on the biochemical properties of the various oxygenases. In this study, cDNA microarray analysis revealed significant changes in gene expression in enterocytes isolated from the small intestine of intestinal epithelium-specific Por knock-out (named IE-Cpr-null) mice compared with that observed in wild-type (WT) littermates. Gene ontology analyses revealed significant changes in terms related to P450s, transporters, cholesterol biosynthesis, and, unexpectedly, antigen presentation/processing. The genomic changes were confirmed at either mRNA or protein level for selected genes, including those of the major histocompatibility complex class II (MHC II). Cholesterol biosynthetic activity was greatly reduced in the enterocytes of the IE-Cpr-null mice, as evidenced by the accumulation of the lanosterol metabolite, 24-dihydrolanosterol. However, no differences in either circulating or enterocyte cholesterol levels were observed between IE-Cpr-null and WT mice. Interestingly, the levels of the cholesterol precursor farnesyl pyrophosphate and its derivative geranylgeranyl pyrophosphate were also increased in the enterocytes of the IE-Cpr-null mice. Furthermore, the expression of STAT1 (signal transducer and activator of transcription 1), a downstream target of geranylgeranyl pyrophosphate signaling, was enhanced. STAT1 is an activator of CIITA, the class II transactivator for MHC II expression; CIITA expression was concomitantly increased in IE-Cpr-null mice. Overall, these findings provide a novel and mechanistic link between POR-dependent enzymes and the expression of MHC II genes in the small intestine.

Biodegradation of 2,4,6‐TCA by the white‐rot fungus Phlebia radiata is initiated by a phase I (O‐demethylation)–phase II (O‐conjugation) reactions system: implications for the chlorine cycle

Thirteen species of white-rot fungi tested have been shown to efficiently biodegrade 1 mM 2,4,6-trichloroanisole (2,4,6-TCA) in liquid cultures. The maximum biodegradation rate (94.5% in 10-day incubations) was exhibited by a Phlebia radiata strain. The enzymes of the ligninolytic complex, laccase, lignin peroxidase (LiP), manganese peroxidase (MnP) and versatile peroxidase (VP) were not able to transform 2,4,6-TCA in in vitro reactions, indicating that the ligninolytic complex was not involved in the initial attack to 2,4,6-TCA. Instead, the first biodegradative steps were carried out by a phase I and phase II reactions system. Phase I reaction consisted on a O-demethylation catalysed by a microsomal cytochrome P-450 monooxygenase to produce 2,4,6-trichlorophenol (2,4,6-TCP). Later, in a phase II reaction catalysed by a microsomal UDP-glucosyltransferase, 2,4,6-TCP was detoxified by O-conjugation with D-glucose to produce 2,4,6-TCP-1-O-d-glucoside (TCPG). This compound accumulated in culture supernatants, reaching its maximum concentration between 48 and 72 h of growth. TCPG levels decreased constantly by the end of fermentation, indicating that it was subsequently metabolized. A catalase activity was able to break in vitro the glycosidic link to produce 2,4,6-TCP, whereas ligninolytic enzymes did not have a significant effect on the biotransformation of that compound. Once formed, 2,4,6-TCP was further degraded as detected by a concomitant release of 2.6 mol of chloride ions by 1 mol of initial 2,4,6-TCA, indicating that this compound underwent almost a complete dehalogenation and biodegradation. It was concluded that P. radiata combines two different degradative mechanisms in order to biodegrade 2,4,6-TCA. The significance of the capability of white-rot fungi to O-demethylate chloroanisoles for the global chlorine cycle is discussed.

The Challenges of Dealing with Promiscuous Drug-Metabolizing Enzymes, Receptors and Transporters

Unlike classical enzymes, drug-metabolizing enzymes (DMEs), such as the liver microsomal cytochrome P450, UDP-glucuronyltransferase, epoxide hydrolase, and flavin-containing monooxygenase, all exhibit broad substrate specificities, low turnover rates, atypical kinetics, and other unusual properties. Receptors (the pregnane X receptor, NR1I2; the constitutive androstane receptor, NR1I3; and the aromatic hydrocarbon receptor) responsible for the induction of DMEs and transporters (P-glycoprotein) responsible for drug transport also have broad substrate specificities. These promiscuous proteins are all intimately involved in drug disposition. Promiscuous proteins, by definition, are known for diversity, but not specificity, in their interaction with drugs. In this review, we analyzed recent advances on the three dimensional structures and kinetic properties of DMD proteins from crystallography, mutational, and kinetic studies to gain insights into the structural and biochemical basis for the promiscuous ligand-protein interactions of the proteins. Large substrate-binding cavities (SBCs), binding of more than one substrate/effector and binding of substrates in alternative orientations and locations within the SBCs, rotation of a substrate at the active site, and substantial substrate-induced conformational changes of the SBCs are common features of the promiscuous DMEs, receptors, and transporters, and therefore, are important parameters to be considered in dealing with drug metabolism issues and safety evaluation of drugs and environmental chemicals.

P450 oxidoreductase: genetic polymorphisms and implications for drug metabolism and toxicity

Background: Cytochrome P450 oxidoreductase (POR) is the only electron donor for all microsomal cytochrome P450 monooxygenases (CYP), some of which are phase I drug-metabolizing enzymes, responsible for oxidation of more than 80% of drugs. Objectives: To provide a more thorough understanding of the genetic factors influencing drug metabolism, we address the role of genetic polymorphisms in the POR gene, and their implications for drug metabolism and cytotoxicity. Methods: The scope of this review is intended to cover polymorphisms currently identified in the POR gene, assess their functional significance on POR activity, and address their impact on CYP-mediated drug metabolism. POR is also responsible for directly metabolizing several anticancer prodrugs via a 1-electron reduction reaction, so the effect of POR polymorphisms on the direct bioactivation of drugs is also considered. Results/conclusion: POR is a polymorphic enzyme that can affect CYP-mediated drug metabolism as well as direct bioactivation of prodrugs. Genetic polymorphisms in the POR gene may help to explain altered drug-metabolizing phenotypes.

Identification of a Biosynthetic Gene Cluster in Rice for Momilactones

Rice diterpenoid phytoalexins such as momilactones and phytocassanes are produced in suspension-cultured rice cells treated with a chitin oligosaccharide elicitor and in rice leaves irradiated with UV light. The common substrate geranylgeranyl diphosphate is converted into diterpene hydrocarbon precursors via a two-step sequential cyclization and then into the bioactive phytoalexins via several oxidation steps. It has been suggested that microsomal cytochrome P-450 monooxygenases (P-450s) are involved in the downstream oxidation of the diterpene hydrocarbons leading to the phytoalexins and that a dehydrogenase is involved in momilactone biosynthesis. However, none of the enzymes involved in the downstream oxidation of the diterpene hydrocarbons have been identified. In this study, we found that a putative dehydrogenase gene (AK103462) and two functionally unknown P-450 genes (CYP99A2 and CYP99A3) form a chitin oligosaccharide elicitor- and UV-inducible gene cluster, together with OsKS4 and OsCyc1, the diterpene cyclase genes involved in momilactone biosynthesis. Functional analysis by heterologous expression in Escherichia coli followed by enzyme assays demonstrated that the AK103462 protein catalyzes the conversion of 3beta-hydroxy-9betaH-pimara-7,15-dien-19,6beta-olide into momilactone A. The double knockdown of CYP99A2 and CYP99A3 specifically suppressed the elicitor-inducible production of momilactones, strongly suggesting that CYP99A2, CYP99A3, or both are involved in momilactone biosynthesis. These results provide strong evidence for the presence on chromosome 4 of a gene cluster involved in momilactone biosynthesis.

Multiple molecular dynamics simulations of human p450 monooxygenase CYP2C9: The molecular basis of substrate binding and regioselectivity toward warfarin

To examine the molecular basis of activity and regioselectivity of the clinically important human microsomal cytochrome P450 (CYP) monooxygenase 2C9 toward its substrate warfarin, 22 molecular dynamics simulations (3-5 ns each) were performed in the presence and absence of warfarin. The resulting trajectories revealed a stable protein core and mobile surface elements. This mobility leads to the formation of two surface channels in the region between F-G loop, B' helix/B-B' loop, beta(1)-sheet, and between helices F and I and the turn in the C-terminal antiparallel beta-sheet in the presence of warfarin. Besides the nonproductive state of the CYP2C9 warfarin complex captured in the crystal structure, three additional states were observed. These states differ in the shape of the substrate binding cavity and the position of the warfarin molecule relative to heme. In one of these states, the 7- and 6-positions of warfarin contact the heme with a marked geometrical preference for position 7 over position 6. This modeling result is consistent with experimentally determined regioselectivity (71 and 22% hydroxylation in positions 7 and 6, respectively). Access to the heme group is limited by the core amino acids Ala297, Leu362, Leu366, and Thr301, which therefore are expected to have a major impact on regioselectivity. In addition, modeling predicts that autoactivation of warfarin is sterically hindered. Our study demonstrates how the combination of mobile surface and rigid core leads to interesting properties: a broad substrate profile and simultaneously a high regioselectivity.

Engineering a Hyper‐Fluorescent NADPH:Cytochrome P450 Oxidoreductase

In order to study molecular interactions in the microsomal cytochrome P450 monooxygenase pathway, we have undertaken the development of a NADPH:cytochrome P450 oxidoreductase (CYPOR) with highly enhanced flavin fluorescence while maintaining wild-type (WT) structure and function. Using a bacterial expression system, a soluble human CYPOR lacking the N-terminal 66 residues was produced, comprised of the catalytic FMN-, FAD- and NADPH-binding domains. Using site-directed mutagenesis of our expression plasmid, we have created six variants of human CYPOR; three having substitutions in the FMN-binding domain (Y143F, Y143A, and Y143S) and three in the FAD-binding domain (Y459F, Y459A, and Y459S). All six variants, as well as WT, were expressed and purified to homogeneity. Flavin analysis showed that Y143 variants had slightly decreased FMN content, while Y459 variants had dramatically reduced FAD content, compared to WT. Y143A and Y143S variants exhibited 3.5- and 6.5-fold increases in flavin fluorescence compared to WT, respectively. The Y143S variant retained 85% of WT NADPH:cytochrome c reductase activity, while Y143A was 70% more active than WT. Based on the observed increase in fluorescence and on the retained catalytic efficiency, Y143 variants currently represent the best candidates for development as tools for studying CYPOR interactions with P450s and other microsomal proteins. With proof of principal established, future constructs will incorporate the membrane anchoring residues, for reconstitution of cytochrome P450-mediated activities in vitro and in cellular expression systems (Supported by NIH HL30050 to BSM).

A Second FMN Binding Site in Yeast NADPH-Cytochrome P450 Reductase Suggests a Mechanism of Electron Transfer by Diflavin Reductases

NADPH-cytochrome P450 reductase transfers two reducing equivalents derived from a hydride ion of NADPH via FAD and FMN to the large family of microsomal cytochrome P450 monooxygenases in one-electron transfer steps. The mechanism of electron transfer by diflavin reductases remains elusive and controversial. Here, we determined the crystal structure of truncated yeast NADPH-cytochrome P450 reductase, which is functionally active toward its physiological substrate cytochrome P450, and discovered a second FMN binding site at the interface of the connecting and FMN binding domains. The two FMN binding sites have different accessibilities to the bulk solvent and different amino acid environments, suggesting stabilization of different electronic structures of the reduced flavin. Since only one FMN cofactor is required for function, a hypothetical mechanism of electron transfer is discussed that proposes shuttling of a single FMN between these two sites coupled with the transition between two semiquinone forms, neutral (blue) and anionic (red).

Recombinant Production of Human Microsomal Cytochrome P450 2D6 in the Methylotrophic Yeast Pichia pastoris

Microsomal cytochrome P450 monooxygenases of groups 1-3 are mainly expressed in the liver and play a crucial role in phase 1 reactions of xenobiotic metabolism. The cDNAs encoding human CYP2D6 and human NADPH-P450 oxidoreductase (CPR) were transformed into the methylotrophic yeast Pichia pastoris and expressed with control of the methanol-inducible AOX1 promoter. The determined molecular weights of the recombinant CYP2D6 and CPR closely matched the calculated values of 55.8 and 76.6 kDa. CPR activity was detected by conversion of cytochrome c by using isolated microsomes. Nearly all of the recombinant CYP was composed of the active holoenzyme, as confirmed by reduced CO difference spectra, which showed a single peak at 450 nm. Only by coexpression of human CPR and CYP was CYP2D6 activity obtained. Microsomes containing human CPR and CYP2D6 converted different substrates, such as 3-cyano-7-ethoxycoumarin, parathion and dextrometorphan. The kinetic parameters of dextrometorphan conversion closely matched those of CYP2D6 from other recombinant expression systems and human microsomes. The endogenous NADPH-P450 oxidoreductase of Pichia pastoris seems to be incompatible with human CYP2D6, as expression of CYP2D6 without human CPR did not result in any CYP activity. These recombinant strains provide a novel, easy-to-handle and cheap source for the biochemical characterisation of single microsomal cytochromes, as well as their allelic variants.