The long term aim of the research described in this proposal is to understand the structural basis for the unique substrate specificities of the human cytochrome P450 family 4 enzymes. CYP4A and 4F enzymes play important roles in the thermodynamically disfavored co-oxidation of endogenous fatty acids such as arachidonic acid and the leukotrienes. Versatile CYP4B enzymes, which are the focus of this proposal, are associated with the bioactivation of a diverse array of pro-toxins including carcinogenic aromatic amines and pneumotoxic furans, while also maintaining to-hydroxylase selectivity for fatty acids and alkyl hydrocarbons. The latter activity may be associated with, as yet, unknown physiological substrate(s) for the enzyme. Human CYP4B1, on the other hand, is an enigmatic enzyme that may have its catalytic function compromised by unique coding region sequences relative to animal forms of the enzyme. These substantial species-differences in metabolism complicate risk assessment in humans for CYP4B1-dependent bioactivation of pro-toxins. To directly address the catalytic function of CYP4B1, and to accommodate the difficulties inherent in working with the human form of the enzyme in heterologous expression systems, we will develop knockout and transgenic mouse models with which to test enzyme function. Specifically, Aim 1 proposes to complete development of a CYP4b1-null mouse line which will be used in Aim 2 to determine the contribution of Cyp4b1 to arylamine induced liver and bladder toxicity.
These Aims will test the hypothesis that CYP4b1, rather than CYPla2 - which has been ruled out as a causative enzyme in previous knockout experiments - is responsible for arylamine bioactivation in the mouse.
Aim 3 will extend the mouse model work to develop a transgenic line carrying various versions of the human CYP4B1gene. This in vivo model is ideally suited to providing an optimal in situ environment for correct folding and heme incorporation of the human enzyme.
Aims 4 and 5 propose to develop an increasingly sophisticated picture of the CYP4B1 active site.
In Aim 4. we will exploit tight binding of hydrocarbons to CYP4B1 to evaluate the dimensions of the active site pocket by spectrophotometric analysis.
In Aim 5 we will capitalize further on this phenomenon by engineering conformationally stabilized hydrocarbon-bound, soluble, monodispersed forms of CYP4B1 suitable for crystallization. The parallel pursuits involving gene-targeted mice and the two-tiered approach to mapping the enzyme's active site are complementary to our overarching goal of understanding structure-function relationships for CYP4B1 at the molecular level.
|Hsu, Mei-Hui; Baer, Brian R; Rettie, Allan E et al. (2017) The Crystal Structure of Cytochrome P450 4B1 (CYP4B1) Monooxygenase Complexed with Octane Discloses Several Structural Adaptations for ?-Hydroxylation. J Biol Chem 292:5610-5621|
|Parkinson, Oliver T; Teitelbaum, Aaron M; Whittington, Dale et al. (2016) Species Differences in Microsomal Oxidation and Glucuronidation of 4-Ipomeanol: Relationship to Target Organ Toxicity. Drug Metab Dispos 44:1598-602|
|Schmidt, Eva M; Wiek, Constanze; Parkinson, Oliver T et al. (2015) Characterization of an Additional Splice Acceptor Site Introduced into CYP4B1 in Hominoidae during Evolution. PLoS One 10:e0137110|
|Wiek, Constanze; Schmidt, Eva M; Roellecke, Katharina et al. (2015) Identification of amino acid determinants in CYP4B1 for optimal catalytic processing of 4-ipomeanol. Biochem J 465:103-14|
|Lockhart, Catherine M; Nakano, Mariko; Rettie, Allan E et al. (2014) Generation and characterization of a murine model of Bietti crystalline dystrophy. Invest Ophthalmol Vis Sci 55:5572-81|
|Nakano, Mariko; Lockhart, Catherine M; Kelly, Edward J et al. (2014) Ocular cytochrome P450s and transporters: roles in disease and endobiotic and xenobiotic disposition. Drug Metab Rev 46:247-60|
|Edson, Katheryne Z; Rettie, Allan E (2013) CYP4 enzymes as potential drug targets: focus on enzyme multiplicity, inducers and inhibitors, and therapeutic modulation of 20-hydroxyeicosatetraenoic acid (20-HETE) synthase and fatty acid ?-hydroxylase activities. Curr Top Med Chem 13:1429-40|
|Parkinson, Oliver T; Liggitt, H Denny; Rettie, Allan E et al. (2013) Generation and characterization of a Cyp4b1 null mouse and the role of CYP4B1 in the activation and toxicity of Ipomeanol. Toxicol Sci 134:243-50|
|Nakano, Mariko; Kelly, Edward J; Wiek, Constanze et al. (2012) CYP4V2 in Bietti's crystalline dystrophy: ocular localization, metabolism of ?-3-polyunsaturated fatty acids, and functional deficit of the p.H331P variant. Mol Pharmacol 82:679-86|
|Parkinson, O T; Kelly, E J; Bezabih, E et al. (2012) Bioactivation of 4-Ipomeanol by a CYP4B enzyme in bovine lung and inhibition by HET0016. J Vet Pharmacol Ther 35:402-5|
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