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 CYP4B1 gene. 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 2-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. ? ? ? ? ?
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