In mammals, cytochrome P450 17A1 catalyzes the 17a-hydroxylation of progesterone and pregnenolone and a second oxidation, the 17a, 20-lyase reaction, to yield androstenedione (Andro) or dihydroepiandrosterone (DHEA).  In teleost fish, there are two enzymes, P450 17A1 (which behaves like the mammalian P450 17A1) and P450 17A2 (which does not catalyze the lyase reaction). Steady-state and some pre-equilibrium kinetics of these reactions have been characterized using recombinant zebrafish proteins.  Binding constants have been determined (spectral titrations) for all P450 17A1 and 17A2 substrates and products. The 2-step P450 17A1 reaction is distributive, as shown by pulse chase experiments, but the pregnenolone reaction is more processive than the progesterone reaction. The results have been fit to a global model.  Although P450 17A2 does not catalyze the oxidation of 17a-hydroxy pregnenolone/ progesterone to DHEA/Andro, a new oxidation product of 17a-hydroxy pregnenolone has been identified.  In order to gain insight into the distinct activities of the zebrafish 17A1 and 17A2 P450s, X-ray crystal structures of both enzymes have been obtained.  Structures of P450 17A1 with abiratrone bound and those of 17A2 in complex with either progesterone or abiraterone were determined at resolutions ranging from 2.8 to 3.3 �. Comparisons of the active site configurations and roles of individual residues in the catalyzed reactions are underway. (Supported in part by USPHS R01 GM103937)

Molecular basis of different steroid oxidation activities in cytochrome P450 17A enzymes

248th National Meeting (2014)

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