title: Active site proton delivery and the lyase activity of human CYP17A1.
abstract: Cytochrome P450 CYP17A1 catalyzes a series of reactions that lie at the intersection of corticoid and androgen biosynthesis and thus occupies an essential role in steroid hormone metabolism. This multifunctional enzyme catalyzes the 17α-hydroxylation of Δ4- and Δ5-steroids progesterone and pregnenolone to form the corresponding 17α-hydroxy products through its hydroxylase activity, and a subsequent 17,20-carbon-carbon scission of pregnene-side chain produce the androgens androstenedione (AD) and dehydroepiandrosterone (DHEA). While the former hydroxylation reaction is believed to proceed through a conventional "Compound I" rebound mechanism, it has been suggested that the latter carbon cleavage is initiated by an iron-peroxy intermediate. We report on the role of Thr306 in CYP17 catalysis. Thr306 is a member of the conserved acid/alcohol pair thought to be essential for the efficient delivery of protons required for hydroperoxoanion heterolysis and formation of Compound I in the cytochromes P450. Wild type and T306A CYP17A1 self-assembled in Nanodiscs were used to quantitate turnover and coupling efficiencies of CYP17's physiological Δ4- and Δ5-substrates. We observed that T306A co-incorporated in Nanodiscs with its redox partner cytochrome P450 oxidoreductase, coupled NADPH only by 0.9% and 0.7% compared to the wild type (97% and 22%) during the conversion of pregnenolone and progesterone, respectively, to the corresponding 17-OH products. Despite increased oxidation of pyridine nucleotide, hydroxylase activity was drastically diminished in the T306A mutant, suggesting a high degree of uncoupling in which reducing equivalents and protons are funneled into non-productive pathways. This is similar to previous work with other P450 catalyzed hydroxylation. However, catalysis of carbon-carbon bond scission by the T306A mutant was largely unimpeded by disruption of the CYP17A1 acid-alcohol pair. The unique response of CYP17A1 lyase activity to mutation of Thr306 is consistent with a reactive intermediate formed independently of proton delivery in the active site, and supports involvement of a nucleophilic peroxo-anion rather than the traditional Compound I in catalysis.
authors.affiliation: Department of Biochemistry, University of Illinois at Urbana Champaign, 116 Morrill Hall, 505 S. Goodwin Avenue, Urbana, IL 61801, United States.
authors.forename: Yogan
authors.initials: Y
authors.lastname: Khatri
authors.affiliation: Department of Biochemistry, University of Illinois at Urbana Champaign, 116 Morrill Hall, 505 S. Goodwin Avenue, Urbana, IL 61801, United States.
authors.forename: Michael C
authors.initials: MC
authors.lastname: Gregory
authors.affiliation: Department of Biochemistry, University of Illinois at Urbana Champaign, 116 Morrill Hall, 505 S. Goodwin Avenue, Urbana, IL 61801, United States.
authors.forename: Yelena V
authors.initials: YV
authors.lastname: Grinkova
authors.affiliation: Department of Biochemistry, University of Illinois at Urbana Champaign, 116 Morrill Hall, 505 S. Goodwin Avenue, Urbana, IL 61801, United States.
authors.forename: Ilia G
authors.initials: IG
authors.lastname: Denisov
authors.affiliation: Department of Biochemistry, University of Illinois at Urbana Champaign, 116 Morrill Hall, 505 S. Goodwin Avenue, Urbana, IL 61801, United States. Electronic address: s-sligar@illinois.edu.
authors.forename: Stephen G
authors.initials: SG
authors.lastname: Sligar
mesh_terms: D002384:Catalysis; D020134:Catalytic Domain; D006801:Humans; D009154:Mutation; D011284:Pregnenolone / Q000737:chemistry / Q000378:metabolism; D011374:Progesterone / Q000737:chemistry / Q000378:metabolism; D011522:Protons; D013254:Steroid 17-alpha-Hydroxylase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013912:Threonine / Q000737:chemistry* / Q000235:genetics
chemical_list: D011522:Protons; D013912:Threonine; D011374:Progesterone; D011284:Pregnenolone; C501892:CYP17A1 protein, human; D013254:Steroid 17-alpha-Hydroxylase
keywords: 17α-hydroxy-pregnenolone; 17α-hydroxy-progesterone; AD; CYP17A1; Compound I; Cpd I; DHEA; Nucelophilic attack; OH-PREG; OH-PROG; PREG; PROG; Proton delivery; T306A; androstenedione; cyt-b(5); cytochrome b(5); dehydroepiandrosterone; pregnenolone; progesterone