Mechanistic Analysis of 17, 20-Lyase Activity for Cytochrome P450c17 Identifies That Cytochrome b5 Influences the Rate of Electron Transfer

Presentation Number: THR-118
Date of Presentation: March 5th, 2015

Alexandr Simonov1, Jessica Holien2, Joyee Yeung1, Ann Dung Ahn Nguyen3, C. Jo Corbin4, Jie Zheng5, Vladimir Kuznetsov6, Richard J. Auchus7, Alan James Conley8, Michael Parker2, Raymond J Rodgers9 and Lisandra Martin*1
1Monash University, 2University of Melbourne, 3UC Davis, Moraga, CA, 4UC Davis, Davis, CA, 5UC Davis, 6Boreskov Institute of Catalysis, 7University of Michigan, Ann Arbor, MI, 8Univ of CA at Davis, Davis, CA, 9University of Adelaide, Adelaide, Australia

Abstract

The steroidogenic enzyme cytochrome P450 17α-hydroxlase (P450c17; encoded by CYP17) is the enzyme needed to synthesize both androgens (male hormones) and glucocorticoids (e.g. cortisol, essential for metabolism). P450c17 has a single enzymatic activity, 17a-hydroxylase, in the zona fascicularis of the adrenal gland which produces cortisol.  However, it has two activities, 17a-hydroxylase and 17,20 lyase, in the gonads and adrenal zona reticularis and these two enzymatic activities are essential for the conversion of pregnenolone to the androgen precursor dehydroepiandrostenedione (DHEA). The first enzymatic activity, 17a-hydroxylase, is a single hydroxylation and the second enzymatic reaction requires an additional hydroxylation leading to scission of the 17,20 carbon bond. Therefore, both activities of P450c17 require electrons from NADPH delivered via cytochrome P450 reductase (CPR). However, 17,20-lyase activity is regulated (enhanced) by a non redox-dependent interaction with cytochrome b5 (cyt b5).

We have used a combination of in vivo, in vitro and in silico approaches to discover then mechanistic function for the regulation of P450c17 activity, by protein-protein interaction, with cyt b5. We provide in silico analysis to confirm the allosteric binding site and disruption of this interface by the double mutant E48G,E49G cyt b5. In live cells we demonstrated using FRET analyses that P450c17 interacts tightly with cyt b5. Using a quartz crystal microbalance analysis we created an artificial lipid membrane and introduced purified proteins which bound and showed that the cyt b5, but not the E48G,E49G cyt b5, formed a complex with the P450c17 and CPR. Finally we employed protein electrochemistry and revealed that P450c17 and cyt b5, but not P450c17 and E48G,E49G cyt b5, were electronically coupled. We identified that the kinetics of electron transfer by P450c17 was slower when the cyt b5 was present and suggest that this may be part of the mechanism that substantially enhances the 17,20 lyase activity of P450c17.

 

Nothing to Disclose: AS, JH, JY, ADAN, CJC, JZ, VK, RJA, AJC, MP, RJR, LM