Activation of androgen biosynthesis and drug metabolism by cytochrome b5

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All 19-carbon androgens derive from 21-carbon steroids via sequential 17¿-hydroxylase and 17,20-lyase activities of cytochrome P450c17 (CYP17A1). The complex chemistry of the 17,20-lyase reaction is selectively stimulated up to 10-fold by cytochrome b5 (b5) in vitro. In contrast to the interactions of b5 with some other cytochromes P450, which apparently involve electron transfer from b5, our data argue that b5 allosterically activates the 17,20-lyase activity of CYP17A1. We have identified a specific region of b5 that is critical for stimulation of 17,20-lyase activity and have shown that the magnitude of this stimulation is substrate-dependent. We now propose to elucidate the importance of this action of b5 on 17,20-lyase activity and androgen synthesis in vivo and to compare the mechanistic and structural features of the b5-CYP17A1 interaction with b5 action on other P450-mediated reactions, in preparation for a detailed interrogation of the mechanism of b5 action on CYP17A1. To first prove that b5 is essential for androgen biosynthesis, we will generate and characterize mice lacking b5 in the testis for Aim 1. We will characterize the fertility, hormone production, and testicular histology of these mice. The enzymatic activity of the testes will be studied in detail. In Aim 2, we will determine if residues on b5 that are required for stimulating 17,20-lyase activity are also essential for modulating the activities of other cytochromes P450 and begin to determine whether the same microscopic steps are involved for all P450 enzymes. We thus will define the importance of b5 action on CYP17A1 in mammalian physiology, and we will begin to ascertain if the mechanism of action is similar to b5 action on other cytochromes P450. This knowledge will provide a novel approach for suppressing androgen production, by targeting the CYP17A1-b5 interaction.