We investigated the regulation mechanisms and the structure-activity relationship of the sex-specific mouse P450s: the male-specific steroid 16- alpha-hydroxylase (P450(16)-alpha) and the female-specific steroid 15- alpha-hydroxylase (P450(15)-alpha). The sex-specific expressions of these P450 genes are regulated by growth hormone transcriptionally. The P450(16)-alpha/CAT recombinant-containing transgenic mice were produced and exhibited the male-specific expression of CAT gene in the livers. The results indicate that the 5.5 kbp 5'-flanking region contains the regulatory elements necessary for the sex-specific transcription of this gene. Using in vitro transcription and transient transfection assays, we identified in the flanking region a cis-acting transcription element, Sex Difference Information (SDI), specific to the P450(16)-alpha gene. Furthermore, our gel-retardation assays showed the presence of nuclear protein which specifically binds to the SDI. For studying the regulation of P450(15)-alpha gene transcription, we identified the genetic variants of inbred mice in which the gene is regulated differently. Our site-directed mutagenesis studies of two highly homologous P450(15)-alpha and P450coh showed that both P450(15)-alpha-dependent 15-alpha-hydroxylase and P450coh-dependent coumarin 7-hydroxylase activities depend critically on the residue at position 209. Our spectral studies of a series of the mutated enzymes suggested that residue-209 resides close to the 6th axial ligand on the distal surface of heme in the P450s and, therefore, plays a critical role in determining the specificity and activity of hydroxylases. The enzyme-kinetic studies showed that residue-209 functions as an important site in the P450's substrate-binding site. Moreover, the size of residue-209 determined the Km, Vmax, Kd and Ki values of the hydroxylases. These results provided the significant advance in understanding the structural basis of P450 reactions. Also, this progress in our research is extremely important to understand the sex- and tissue-dependent toxicity and carcinogenicity of endogenous hormones and exogenous chemicals and their polymorphism.
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