Design of a novel P450: a functional bacterial-human cytochrome P450 chimera

We report the construction of a functional chimera from approximately 50% bacterial (cytosolic) cytochrome P450cam and 50% mammalian (membrane-bound) cytochrome P450 2C9. The chimeric protein shows a reduced CO-difference spectrum absorption at 446 nm, and circular dichroism spectra indicate that the protein is globular. The protein is soluble and catalyzes the oxidation of 4-chlorotoluene using molecular oxygen and reducing equivalents from bacterial putidaredoxin and putidaredoxin reductase. This chimera provides a novel method for addressing structure-function issues and may prove useful in the design of oxidants for benign and stereospecific synthesis, as well as catalysts for bioremediation of polluted areas. Furthermore, these results provide the first evidence that bacterial P450 enzymes and mammalian P450 enzymes are likely to share a common tertiary structure.     

Interactions of substrate and product with cytochrome P450: P4502B4 versus P450cam

OBJECTIVE: To determine the relative abilities of somatostatin receptor scintigraphy (SRS) and conventional imaging studies (computed tomography, magnetic resonance imaging, ultrasound, angiography) to localize gastrinomas before surgery in patients with Zollinger-Ellison syndrome (ZES) subsequently found at surgery, and to determine the effect of SRS on the disease-free rate. SUMMARY BACKGROUND DATA: Recent studies demonstrate that SRS is the most sensitive imaging modality for localizing neuroendocrine tumors such as gastrinomas. Because of conflicting results in small series, it is unclear in ZES whether SRS will alter the disease-free rate, which gastrinomas are not detected, what factors contribute to failure to detect a gastrinoma, or whether the SRS result should be used to determine operability in patients without hepatic metastases, as recently recommended by some investigators. METHODS: Thirty-five consecutive patients with ZES undergoing 37 exploratory laparotomies for possible cure were prospectively studied. All had SRS and conventional imaging studies before surgery. Imaging results were determined by an independent investigator depending on surgical findings. All patients underwent an identical surgical protocol (palpation after an extensive Kocher maneuver, ultrasound during surgery, duodenal transillumination, and 3 cm duodenotomy) and postoperative assessment of disease status (fasting gastrin, secretin test imaging within 2 weeks, at 3 to 6 months, and yearly), as used in pre-SRS studies previously. RESULTS: Gastrinomas were detected in all patients at each surgery. Seventy-four gastrinomas were found: 22 duodenal, 8 pancreatic, 3 primaries in other sites, and 41 lymph node metastases. The relative detection order on a per-patient or per-lesion basis was SRS > angiography, magnetic resonance imaging, computed tomography > ultrasound. On a per-lesion basis, SRS had greater sensitivity than all conventional studies combined. SRS missed one third of all lesions found at surgery. SRS detected 30% of gastrinomas < or =1.1 cm, 64% of those 1.1 to 2 cm, and 96% of those >2 cm and missed primarily small duodenal tumors. Tumor size correlated closely with SRS rate of detection. SRS did not increase the disease-free rate immediately after surgery or at 2 years mean follow-up. CONCLUSIONS: SRS is the most sensitive preoperative imaging study for extrahepatic gastrinomas in patients with ZES and should replace conventional imaging studies as the preoperative study of choice. Negative results of SRS for localizing extrahepatic gastrinomas should not be used to decide operability, because a surgical procedure will detect 33% more gastrinomas than SRS. SRS does not increase the disease-free rate. In the future, more sensitive methods to detect small gastrinomas, especially in the duodenum and in periduodenal lymph nodes, or more extensive surgery will be needed to improve the postoperative disease-free rate in ZES.     

Avian forms of cytochrome P450

Despite the importance of avian P450 forms in modulation of the toxicity of pesticides and other environmental chemicals, relatively little work has been done upon them, and very few forms have been fully characterised. An avian form that appears to belong to family 1A is readily inducible by planar molecules (e.g. coplanar PCB's, PCDD and certain PAHs) and has been the basis of a biomarker assay used in field studies. Although it is recognised by antibodies for mammalian P450 1A1, it evidently differs from the mammalian forms of the enzyme in catalytic properties. Phenobarbitone induces two forms of P450 in the domestic fowl (2H1 and 2H2) which have been purified, and these resemble P450 2B1 and P450 2B2 of the rat respectively. Two further phenobarbitone inducible forms, PB-A and PB-B have been partially purified. Also there is an acetone inducible form that resembles rodent P450 2E. In field studies evidence has been produced for the induction of P450s recognised by antibodies to mammalian forms of P450 1A1 and P450 2B in avian liver (adults and embryos), in response to environmental levels of PCBs. Fungicides which act as ergosterol biosynthesis inhibitors (EBI fungicides) such as prochloraz and propiconazole potentiate the toxicity of certain phosphorothionates to birds.     

Physiological and pathophysiological regulation of cytochrome P450

This article is a report on a symposium sponsored by the American Society for Pharmacology and Experimental Therapeutics and held at the April 1998 Experimental Biology '98 meeting in San Francisco. The presentations focused on the mechanisms of regulation of cytochrome P450 gene expression by developmental factors and by hormones and cytokines, as well as on the interplay between physiological and chemical regulation. Approaches and systems used to address these questions included conditional gene knockouts in mice, primary hepatocyte cultures, immunofluorescence imaging of cells, and cell lines stably expressing reporter gene constructs.     

Specificity of self-assembly of cytochrome P450

Under certain conditions, hexamers of microsomal cytochrome P450 can self-assemble from the subunits of different isoforms. However, the possibility for free choice results in recognition between identical subunits of each form of cytochrome P450 which provides preferential association of identical monomers into corresponding hexamers. The specificity of self-assembly suggests hexameric arrangement of cytochrome P450 in native membranes as we proposed earlier. In the present study, highly purified cytochrome P450 2B4 and cytochrome P450 1A2 (CYP 2B4 and CYP 1A2), including those immobilized by covalent attachment to an insoluble carrier of one protomer of each hexamer, were employed.     

Reconstruction of mitochondrial cytochrome P450-dependent steroid hydroxylases in artificial phospholipid membranes: studies of cytochrome P450scc topology by limited proteolysis

Highly purified cytochrome P450scc from bovine adrenal cortex mitochondria was inserted in artificial phospholipid membranes prepared from phosphatidylcholine to study the main principles of its membrane organization in the model system. Topology of the cytochrome P450scc polypeptide chain in proteoliposomes was studied by limited proteolysis with trypsin or chymotrypsin followed by immunochemical identification of the products of proteolysis products of the membrane-bound heme protein. It is shown that limited proteolysis of cytochrome P450scc in proteoliposomes results in a significant decrease of Vmax for the reaction of cholesterol hydroxylation to pregnenolone in the reconstituted system in the presence of exogenously added adrenodoxin-reductase and adrenodoxin. However, after proteolytic modification of cytochrome P450scc with trypsin and chymotrypsin the affinity of the heme protein to adrenodoxin is increased. Different models of membrane organization as well as functional specificity of cytochrome P450scc in artificial membranes are discussed.     

Inducers of the cytochrome P-450 superfamily as promoters of carcinogenesis

This review summarizes data on the mechanisms of tumor-promoting activity of non-genotoxic compounds that are inducers of cytochrome P-450 isoforms. Their promoting activity is analyzed in term of synthesis of new cytochrome P-450 isoforms. Active oxygen species formed by cytochrome P-450 isoforms can simultaneously act as stimulators of proliferation and inhibitors of intercellular communications. Promoter effects can be associated with changes in the ratio of cellular signaling non-protein molecules induced by newly synthesized cytochrome P-450 isoforms. Data on induction of cytochrome P-450 isoforms of family 1 indicate that inducer interaction with its receptor causes cellular events resulting in the stimulation of cell proliferation.     

Enhanced expression of cytochrome P450 in stomach cancer

The cytochromes P450 have a central role in the oxidative activation and detoxification of a wide range of xenobiotics, including many carcinogens and several anti-cancer drugs. Thus the cytochrome P450 enzyme system has important roles in both tumour development and influencing the response of tumours to chemotherapy. Stomach cancer is one of the commonest tumours of the alimentary tract and environmental factors, including dietary factors, have been implicated in the development of this tumour. This type of tumour has a poor prognosis and responds poorly to current therapies. In this study, the presence and cellular localization of several major forms of P450, CYP1A, CYP2E1 and CYP3A have been investigated in stomach cancer and compared with their expression in normal stomach. There was enhanced expression of CYP1A and CYP3A in stomach cancer with CYP1A present in 51% and CYP3A present in 28% of cases. In contrast, no P450 was identified in normal stomach. The presence of CYP1A and CYP3A in stomach cancer provides further evidence for the enhanced expression of specific forms of cytochrome P450 in tumours and may be important therapeutically for the development of anti-cancer drugs that are activated by these forms of P450.     

Escherichia coli expression and substrate specificities of canine cytochrome P450 3A12 and rabbit cytochrome P450 3A6

High level Escherichia coli expression of cytochromes P450 3A12 and 3A6 has facilitated the characterization of proteins which exhibit limited activity as purified hepatic enzymes in reconstituted systems. Three 3A12 and two 3A6 constructs modified at the 5'-end to encode the bovine 17 alpha-sequence (Barnes et al., Proc. Natl. Acad. Sci. U.S.A. 88: 5597-5601, 1991), or related sequences, exhibited expression levels ranging from 2 to 89 nmol of cytochrome P450 liter-1. Recombinant canine 3A12 catalyzed steroid 6 beta-hydroxylation and erythromycin demethylation at rates comparable to those obtained in phenobarbital-induced canine liver microsomes. In contrast, 3A12 troleandomycin demethylase activity (2.5 nmol/min/nmol) was significantly lower than that of canine phenobarbital-induced liver microsomes (6.6 nmol/min/nmol). This difference in activity suggests that at least two 3A forms, which may differ functionally, are present within the canine liver. Purification of recombinant rabbit 3A6 revealed that homogeneous and E. coli-solubilized membrane preparations of 3A6 exhibit similar metabolic rates and identical substrate specificities; 3A activity was modulated by 25 microM alpha-naphthoflavone, which stimulated an unidentified progesterone metabolite 9-fold in 3A6 reconstituted systems in contrast to the 4-fold stimulation of 3A12. Furthermore, 25 microM alpha-naphthoflavone inhibited erythromycin demethylation 64 and 33% by purified recombinant 3A6- or 3A6-solubilized membrane fractions, respectively; 3A12-mediated erythromycin demethylation in solubilized membrane fractions was resistant to flavonoid inhibition. These results indicate that, although 3A substrate specificities are highly conserved between species, functional differences exist between canine 3A12 and rabbit 3A6, which may be utilized to better understand 3A structure-function relationships.     

The substrate specificity of cytochrome P450cam

The catalytic turnover of xenobiotics by cytochrome P450cam results in both the formation of organic metabolites and the uncoupled production of H2O2, and H2O. Previous studies have shown that a receptor-constrained three-dimensional screening program (DOCK) can be used to identify potential ligands (ergo substrates) for the enzyme (De Voss, J. J.; Sibbesen, O.; Zhang, Z.; Ortiz de Montellano, P. R. J. Am. Chem. Soc. 1997, 119, 5489). A new set of 10 compounds has now been examined to further test the substrate specificity of P450cam and the ability of DOCK to identify substrates for this enzyme. The results expand the known specificity of P450cam and define limitations in the use of DOCK to predict its substrate specificity.     

Evolution of the cytochrome P450 superfamily: sequence alignments and pharmacogenetics

The evolution of the cytochrome P450 (CYP) superfamily is described, with particular reference to major events in the development of biological forms during geological time. It is noted that the currently accepted timescale for the elaboration of the P450 phylogenetic tree exhibits close parallels with the evolution of terrestrial biota. Indeed, the present human P450 complement of xenobiotic-metabolizing enzymes may have originated from coevolutionary 'warfare' between plants and animals during the Devonian period about 400 million years ago. A number of key correspondences between the evolution of P450 system and the course of biological development over time, point to a mechanistic molecular biology of evolution which is consistent with a steady increase in atmospheric oxygenation beginning over 2000 million years ago, whereas dietary changes during more recent geological time may provide one possible explanation for certain species differences in metabolism. Alignment between P450 protein sequences within the same family or subfamily, together with across-family comparisons, aid the rationalization of drug metabolism specificities for different P450 isoforms, and can assist in an understanding of genetic polymorphisms in P450-mediated oxidations at the molecular level. Moreover, the variation in P450 regulatory mechanisms and inducibilities between different mammalian species are likely to have important implications for current procedures of chemical safety evaluation, which rely on pure genetic strains of laboratory bred rodents for the testing of compounds destined for human exposure.     

Evaluation of atypical cytochrome P450 kinetics with two-substrate models: evidence that multiple substrates can simultaneously bind to cytochrome P450 active sites

Some cytochrome P450 catalyzed reactions show atypical kinetics, and these kinetic processes can be grouped into five categories: activation, autoactivation, partial inhibition, substrate inhibition, and biphasic saturation curves. A two-site model in which the enzyme can bind two substrate molecules simultaneously is presented which can be used to describe all of these observed kinetic properties. Sigmoidal kinetic characteristics were observed for carbamazepine metabolism by CYP3A4 and naphthalene metabolism by CYPs 2B6, 2C8, 2C9, and 3A5 as well as dapsone metabolism by CYP2C9. Naphthalene metabolism by CYP3A4 and naproxen metabolism by CYP2C9 demonstrated nonhyperbolic enzyme kinetics suggestive of a low Km, low Vmax component for the first substrate molecule and a high Km, high Vmax component for the second substrate molecule. 7, 8-Benzoflavone activation of phenanthrene metabolism by CYP3A4 and dapsone activation of flurbiprofen and naproxen metabolism by CYP2C9 were also observed. Furthermore, partial inhibition of 7, 8-benzoflavone metabolism by phenanthrene was observed. These results demonstrate that various P450 isoforms may exhibit atypical enzyme kinetics depending on the substrate(s) employed and that these results may be explained by a model which includes simultaneous binding of two substrate molecules in the active site.     

Phytanic acid alpha-hydroxylation by bacterial cytochrome P450

The science of drug metabolism, like any other science, has advanced from simple beginnings (by today's standards) to its present state. One can examine the path that has been taken to understand the forces driving the direction of evolution of this science. The trends discovered can then be used to make reasonable extrapolations about the changes that might be expected in the future. That exercise is the subject of this article. The main focus will be on drug metabolism as practiced in the industrial environment, representing the author's main experience as well as the principal arena of practical applications of the science. The discussion will draw mainly on broad phenomena occurring in this application of drug metabolism to drug discovery and development.     

The domain architecture of cytochrome P450BM-3

Cytochromes P450 utilize redox partners to deliver electrons from NADPH/NADH to the P450 heme center. Microsomal P450s utilize an FAD/FMN reductase. The bacterial fatty acid hydroxylase, P450BM-3, is similar except the P450 heme and FAD/FMN proteins are linked together in a single polypeptide chain arranged as heme-FMN-FAD. Sequence comparisons indicate that the P450BM-3 FMN and FAD domains are similar to flavodoxin and ferredoxin reductase, respectively. Previous work has shown that the heme and FMN/FAD domains can be separately expressed and purified. In this study we have expressed, purified, and characterized the following additional domains: heme-FMN, FMN, and FAD. Each domain retains their prosthetic groups although the FMN domain is more labile. The FAD domain retains a high level of ferricyanide reductase activity but no cytochrome c reductase activity. In addition, we have deleted a 110-residue stretch in the FAD domain that is not present in ferredoxin reductase. This protein retains both FAD and heme but not FMN. We also have investigated the dimerization pattern of the individual domains that lead to the following conclusions. Holo-P450BM-3 appears to dimerize via interactions that do not involve disulfide bond formation, whereas the reductase and FAD domains form intermolecular disulfides. This indicates that the Cys residues not available for dimerization in holo-P450BM-3 are unmasked in the individual domains.     

Significance of hepatic cytochrome P450 enzymes for psychopharmacology

Nearly all psychotropic drugs are metabolized by hepatic cytochrome P450-enzymes. In humans, there are 5 isoenzymes involved in this process. The activity of these enzymes can be modulated by a number of commonly used drugs, yielding potentially hazardous interactions. Most of the recently introduced selective serotonin reuptake inhibitors are potent inhibitors of cytochrome P450 enzymes. Thus, the plasma concentrations of tricyclic antidepressants or clozapine might be elevated into toxic levels. In contrast, carbamazepine induces most of the isoenzymes. This potentiates the elimination of tricyclics and antipsychotics and might cause a serious risk for the recurrence of depressive or psychotic symptoms. Moreover, 5-10% of the population are slow metabolizers of CYP2D6. This group is prone to increased adverse effects of moderately dosed medication. This review systematically points out the reported or predicted pharmacokinetic drug interactions in psychopharmacology focussing on clinical significance.