Mechanism of the Clinically Relevant E305G Mutation in Human P450 CYP17A1.

Steroid metabolism in humans originates from cholesterol and involves several enzyme reactions including dehydrogenation, hydroxylation, and carbon-carbon bond cleavage that occur at regio- and stereo-specific points in the four-membered ring structure. Cytochrome P450s occur at critical junctions that control the production of the male sex hormones (androgens), the female hormones (estrogens) as well as the mineralocorticoids and glucocorticoids. An important branch point in human androgen production is catalyzed by cytochrome P450 CYP17A1 and involves an initial Compound I-mediated hydroxylation at the 17-position of either progesterone (PROG) or pregnenolone (PREG) to form 17-hydroxy derivatives, 17OH-PROG and 17OH-PREG, with approximately similar efficiencies. Subsequent processing of the 17-hydroxy substrates involves a C17-C20 bond scission (lyase) activity that is heavily favored for 17OH-PREG in humans. The mechanism for this lyase reaction has been debated for several decades, some workers favoring a Compound I-mediated process, with others arguing that a ferric peroxo- is the active oxidant. Mutations in CYP17A1 can have profound clinical manifestations. For example, the replacement of the glutamic acid side with a glycine chain at position 305 in the CYP17A1 structure causes a clinically relevant steroidopathy; E305G CYP17A1 displays a dramatic decrease in the production of dehydroepiandrosterone from pregnenolone but surprisingly increases the activity of the enzyme toward the formation of androstenedione from progesterone. To better understand the functional consequences of this mutation, we self-assembled wild-type and the E305G mutant of CYP17A1 into nanodiscs and examined the detailed catalytic mechanism. We measured substrate binding, spin state conversion, and solvent isotope effects in the hydroxylation and lyase pathways for these substrates. Given that, following electron transfer, the ferric peroxo- species is the common intermediate for both mechanisms, we used resonance Raman spectroscopy to monitor the positioning of important hydrogen-bonding interactions of the 17-OH group with the heme-bound peroxide. We discovered that the E305G mutation changes the orientation of the lyase substrate in the active site, which alters a critical hydrogen bonding of the 17-alcohol to the iron-bound peroxide. The observed switch in substrate specificity of the enzyme is consistent with this result if the hydrogen bonding to the proximal peroxo oxygen is necessary for a proposed nucleophilic peroxoanion-mediated mechanism for CYP17A1 in carbon-carbon bond scission.

Structural features and dynamic investigations of the membrane-bound cytochrome P450 17A1.

Cytochrome P450 (CYP) 17A1 is a dual-function monooxygenase with a critical role in the synthesis of many human steroid hormones. The enzyme is an important target for treatment of breast and prostate cancers that proliferate in response to estrogens and androgens. Despite the crystallographic structures available for CYP17A1, no membrane-bound structural features of this enzyme at atomic level are available. Accumulating evidence has indicated that the interactions between bounded CYPs and membrane could contribute to the recruitment of lipophilic substrates. To this end, we have investigated the effects on structural characteristics in the presence of the membrane for CYP17A1. The MD simulation results demonstrate a spontaneous insertion process of the enzyme to the lipid. Two predominant modes of CYP17A1 in the membrane are captured, characterized by the depths of insertion and orientations of the enzyme to the membrane surface. The measured heme tilt angles show good consistence with experimental data, thereby verifying the validity of the structural models. Moreover, conformational changes induced by the membrane might have impact on the accessibility of the active site to lipophilic substrates. The dynamics of internal aromatic gate formed by Trp220 and Phe224 are suggested to regulate tunnel opening motions. The knowledge of the membrane binding characteristics could guide future experimental and computational works on membrane-bound CYPs so that various investigations of CYPs in their natural, lipid environment rather than in artificially solubilized forms may be achieved.