1. Xenobiotics/steroid metabolizing enzymes, cytochrome P450s and sulfotransferases (STs) exhibit remarkedly diverse substrate and product specificities. Our site-directed mutagenesis studies have shown that a single mutation of the few key amino acid residues is sufficient to alter the specificity. Thus, the substrate pocket is structurally flexible so as to accommodate various chemicals. The flexibility has efficiently fixed """"""""natural"""""""" mutations of a P450 (polymorphism) responsible for many differences in the rate of drug and steroid metabolisms. To determine the 3D structures of P450s and STs, we have now established the bacterial expression system and obtained a large amount of the pure P450s and STs. Moreover, the 2 STs (estrogen and hydroxysteroid sulfotransferases) have been crystallized, diffracting approximately 2-3 A. 2. Some P450 and ST genes are expressed either males or females. The specific expressions are regulated by growth hormone transcriptionally. Very often, P450 genes are also induced by xenobiotics. These sex-specific or xenobiotics-induced expression may result in increasing/decreasing toxic and/or carcinogenic metabolites of xenobiotics. To understand sex-specific transcription, we have purified, cloned and bacterially expressed 2 transcription factors GSBP and NF2d9. GABP binds and activates the CpG demethylated promoter of the male-specific P4502d9 gene. Phenobarbital (PB) is a prototype of many chemicals which induce the same subset of P450 genes. We have established mouse primary hepatocyte system in which PB activates transcription of the endogenous P4502B10 gene and also the various promoter/CAT reporter constructs. Moreover, a 34 bp of PB enhancer element has been identified and a possible binding protein (51 KDa) has been purified.
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