Publications

• Role of cytochrome b5 in the modulation of the enzymatic activities of cytochrome P450 17alpha-hydroxylase/17,20-lyase (P450 17A1).

  Bhatt M.R., Khatri Y., Rodgers R.J., Martin L.L.

  Cytochrome b5 (cyt b5) is a small hemoprotein that plays a significant role in the modulation of activities of an important steroidogenic enzyme, cytochrome P450 17α-hydroxylase/17,20-lyase (P450 17A1, CYP17A1). Located in the zona fasciculata and zona reticularis of the adrenal cortex and in the ... >> More

  Cytochrome b5 (cyt b5) is a small hemoprotein that plays a significant role in the modulation of activities of an important steroidogenic enzyme, cytochrome P450 17α-hydroxylase/17,20-lyase (P450 17A1, CYP17A1). Located in the zona fasciculata and zona reticularis of the adrenal cortex and in the gonads, P450 17A1 catalyzes two different reactions in the steroidogenic pathway; the 17α-hydroxylation and 17,20-lyase, in the endoplasmic reticulum of these respective tissues. The activities of P450 17A1 are regulated by cyt b5 that enhances the 17,20-lyase reaction by promoting the coupling of P450 17A1 and cytochrome P450 reductase (CPR), allosterically. Cyt b5 can also act as an electron donor to enhance the 16-ene-synthase activity of human P450 17A1. In this review, we discuss the many roles of cyt b5 and focus on the modulation of CYP17A1 activities by cyt b5 and the mechanisms involved. << Less

  J Steroid Biochem Mol Biol 170:2-18(2017) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Cytochrome b5 augments the 17,20-lyase activity of human P450c17 without direct electron transfer.

  Auchus R.J., Lee T.C., Miller W.L.

  In the biosynthesis of steroid hormones, P450c17 is the single enzyme that catalyzes both the 17alpha-hydroxylation of 21-carbon steroids and the 17,20-lyase activity that cleaves the C17-C20 bond to produce C19 sex steroids. Cytochrome b5 augments the 17,20-lyase activity of cytochrome P450c17 in ... >> More

  In the biosynthesis of steroid hormones, P450c17 is the single enzyme that catalyzes both the 17alpha-hydroxylation of 21-carbon steroids and the 17,20-lyase activity that cleaves the C17-C20 bond to produce C19 sex steroids. Cytochrome b5 augments the 17,20-lyase activity of cytochrome P450c17 in vitro, but this has not been demonstrated in membranes, and the mechanism of this action is unknown. We expressed human P450c17, human P450-oxidoreductase (OR), and/or human cytochrome b5 in Saccharomyces cerevisiae and analyzed the 17alpha-hydroxylase and 17,20-lyase activities of the resulting yeast microsomes. Yeast expressing only P450c17 have 17alpha-hydroxylase and trace 17,20-lyase activities toward both Delta4 and Delta5 steroids. Coexpression of human OR with P450c17 increases the Vmax of both the 17alpha-hydroxylase and 17,20-lyase reactions 5-fold; coexpression of human b5 with P450c17 also increases the Vmax of the 17,20-lyase reactions but not of the 17alpha-hydroxylase reactions. Simultaneous expression of human b5 with P450c17 and OR, or addition of purified human b5 to microsomes from yeast coexpressing human P450c17 and OR, further increases the Vmax of the 17,20-lyase reaction without altering 17alpha-hydroxylase activity. Genetically engineered yeast and mixing experiments demonstrate that OR is both necessary and sufficient for microsomal 17,20-lyase activity. Addition of purified human holo-b5, apo-b5, or cytochrome c to microsomes containing both human P450c17 and OR demonstrate that the stimulatory action of b5 does not require electron transfer from b5 to P450c17. These data suggest that human b5 acts principally as an allosteric effector that interacts primarily with the P450c17.OR complex to stimulate 17, 20-lyase activity. << Less

  J. Biol. Chem. 273:3158-3165(1998) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.

• Unveiling the crucial intermediates in androgen production.

  Mak P.J., Gregory M.C., Denisov I.G., Sligar S.G., Kincaid J.R.

  Ablation of androgen production through surgery is one strategy against prostate cancer, with the current focus placed on pharmaceutical intervention to restrict androgen synthesis selectively, an endeavor that could benefit from the enhanced understanding of enzymatic mechanisms that derives from ... >> More

  Ablation of androgen production through surgery is one strategy against prostate cancer, with the current focus placed on pharmaceutical intervention to restrict androgen synthesis selectively, an endeavor that could benefit from the enhanced understanding of enzymatic mechanisms that derives from characterization of key reaction intermediates. The multifunctional cytochrome P450 17A1 (CYP17A1) first catalyzes the typical hydroxylation of its primary substrate, pregnenolone (PREG) and then also orchestrates a remarkable C17-C20 bond cleavage (lyase) reaction, converting the 17-hydroxypregnenolone initial product to dehydroepiandrosterone, a process representing the first committed step in the biosynthesis of androgens. Now, we report the capture and structural characterization of intermediates produced during this lyase step: an initial peroxo-anion intermediate, poised for nucleophilic attack on the C20 position by a substrate-associated H-bond, and the crucial ferric peroxo-hemiacetal intermediate that precedes carbon-carbon (C-C) bond cleavage. These studies provide a rare glimpse at the actual structural determinants of a chemical transformation that carries profound physiological consequences. << Less

  Proc Natl Acad Sci U S A 112:15856-15861(2015) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Mechanism of 17alpha,20-Lyase and New Hydroxylation Reactions of Human Cytochrome P450 17A1: 18O LABELING AND OXYGEN SURROGATE EVIDENCE FOR A ROLE OF A PERFERRYL OXYGEN.

  Yoshimoto F.K., Gonzalez E., Auchus R.J., Guengerich F.P.

  Cytochrome P450 (P450) reactions can involve C-C bond cleavage, and several of these are critical in steroid and sterol biosynthesis. The mechanisms of P450s 11A1, 17A1, 19A1, and 51A1 have been controversial, in the context of the role of ferric peroxide (FeO2 (-)) versus perferryl (FeO(3+), comp ... >> More

  Cytochrome P450 (P450) reactions can involve C-C bond cleavage, and several of these are critical in steroid and sterol biosynthesis. The mechanisms of P450s 11A1, 17A1, 19A1, and 51A1 have been controversial, in the context of the role of ferric peroxide (FeO2 (-)) versus perferryl (FeO(3+), compound I) chemistry. We reinvestigated the 17α-hydroxyprogesterone and 17α-hydroxypregnenolone 17α,20-lyase reactions of human P450 17A1 and found incorporation of one (18)O atom (from (18)O2) into acetic acid, consonant with proposals for a ferric peroxide mechanism (Akhtar, M., Lee-Robichaud, P., Akhtar, M. E., and Wright, J. N. (1997) J. Steroid Biochem. Mol. Biol. 61, 127-132; Akhtar, M., Wright, J. N., and Lee-Robichaud, P. (2011) J. Steroid Biochem. Mol. Biol. 125, 2-12). However, the reactions were supported by iodosylbenzene (a precursor of the FeO(3+) species) but not by H2O2 We propose three mechanisms that can involve the FeO(3+) entity and that explain the (18)O label in the acetic acid, two involving the intermediacy of an acetyl radical and one a steroid 17,20-dioxetane. P450 17A1 was found to perform 16-hydroxylation reactions on its 17α-hydroxylated products to yield 16,17α-dihydroxypregnenolone and progesterone, suggesting the presence of an active perferryloxo active species of P450 17A1 when its lyase substrate is bound. The 6β-hydroxylation of 16α,17α-dihydroxyprogesterone and the oxidation of both 16α,17α-dihydroxyprogesterone and 16α,17α-dihydroxypregnenolone to 16-hydroxy lyase products were also observed. We provide evidence for the contribution of a compound I mechanism, although contribution of a ferric peroxide pathway in the 17α,20-lyase reaction cannot be excluded. << Less

  J. Biol. Chem. 291:17143-17164(2016) [PubMed] [EuropePMC]

  This publication is cited by 6 other entries.