title: Unveiling the crucial intermediates in androgen production.
abstract: 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.
authors.affiliation: Department of Chemistry, Marquette University, Milwaukee, WI 53233;
authors.forename: Piotr J
authors.initials: PJ
authors.lastname: Mak
authors.affiliation: Department of Biochemistry, University of Illinois, Urbana, IL 61801;
authors.forename: Michael C
authors.initials: MC
authors.lastname: Gregory
authors.affiliation: Department of Biochemistry, University of Illinois, Urbana, IL 61801;
authors.forename: Ilia G
authors.initials: IG
authors.lastname: Denisov
authors.affiliation: Department of Biochemistry, University of Illinois, Urbana, IL 61801; Department of Chemistry, University of Illinois, Urbana, IL 61801; College of Medicine, University of Illinois, Urbana, IL 61801 s-sligar@illinois.edu james.kincaid@marquette.edu.
authors.forename: Stephen G
authors.initials: SG
authors.lastname: Sligar
authors.affiliation: Department of Chemistry, Marquette University, Milwaukee, WI 53233; s-sligar@illinois.edu james.kincaid@marquette.edu.
authors.forename: James R
authors.initials: JR
authors.lastname: Kincaid
mesh_terms: D006907:17-alpha-Hydroxypregnenolone / Q000737:chemistry / Q000378:metabolism*; D000728:Androgens / Q000737:chemistry / Q000378:metabolism*; D055162:Biocatalysis; D053898:Biosynthetic Pathways; D003687:Dehydroepiandrosterone / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D006860:Hydrogen Bonding; D006900:Hydroxylation; D008956:Models, Chemical; D008958:Models, Molecular; D015394:Molecular Structure; D011284:Pregnenolone / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D013053:Spectrophotometry / Q000379:methods; D013254:Steroid 17-alpha-Hydroxylase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D000728:Androgens; D006907:17-alpha-Hydroxypregnenolone; D003687:Dehydroepiandrosterone; D011284:Pregnenolone; C501892:CYP17A1 protein, human; D013254:Steroid 17-alpha-Hydroxylase
keywords: cytochrome P450; nanodiscs; peroxo-hemiacetal; resonance Raman spectroscopy; steroids