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Cited by 185Metal-Catalyzed Oxygenation MechanismsProtein Structure and DynamicsSteroid Chemistry and Biochemistry

Molecular Modeling of Human P450c17 (17α-Hydroxylase/ 17,20-Lyase): Insights into Reaction Mechanisms and Effects of Mutations

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Auchus, Richard J., Miller, Walter L.·1999·Molecular Endocrinology
DOI 10.1210/mend.13.7.0326
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Abstract

P450c17 (17alpha-hydroxylase/17,20-lyase) catalyzes steroid 17alpha-hydroxylase and 17,20-lyase activities in the biosynthesis of androgens and estrogens. These two activities are differentially regulated in a tissue-specific and developmentally programmed manner. To visualize the active site topology of human P450c17 and to study the structural basis of its substrate specificity and catalytic selectivity, we constructed a second-generation computer-graphic model of human P450c17. The energetics of the model are comparable to those of the principal template of the model, P450BMP, as determined from its crystallographic coordinates. The protein structure analysis programs PROCHECK, WHATIF, and SurVol indicate that the predicted P450c17 structure is reasonable. The hydrophobic active site accommodates both delta4 and delta5 steroid substrates in a catalytically favorable orientation. The predicted contributions of positively charged residues to the redox-partner binding site were confirmed by site-directed mutagenesis. Molecular dynamic simulations with pregnenolone, 17-OH-pregnenolone, progesterone, and 17-OH-progesterone docked into the substrate-binding pocket demonstrated that regioselectivity of the hydroxylation reactions is determined both by proximity of hydrogens to the iron-oxo complex and by the stability of the carbon radicals generated after hydrogen abstraction. The model explains the activities of all known naturally occurring and synthetic human P450c17 mutants. The model predicted that mutation of lysine 89 would disrupt 17,20-lyase activity to a greater extent than 17alpha-hydroxylase activity; expression of a test mutant, K89N, in yeast confirmed this prediction. Hydrogen peroxide did not support catalysis of the 17,20-lyase reaction, as would be predicted by mechanisms involving a ferryl peroxide. Our present model and biochemical data suggest that both the hydroxylase and lyase activities proceed from a common steroid-binding geometry by an iron oxene mechanism. This model will facilitate studies of sex steroid synthesis and its disorders and the design of specific inhibitors useful in chemotherapy of sex steroid-dependent cancers.

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  • The model identifies Arg347 and Arg358 and several other arginine and lysine residues in the redox partner binding site; mutations of these residues all cause varying degrees of selective loss of 17,20-lyase activity.

    “The model identifies Arg 347 and Arg 358 and several other arginine and lysine residues in the redox partner binding site; mutations of these residues all cause varying degrees of selective loss of 17,20lyase activity (240, 279, 280, 284).”

    From The Molecular Biology, Biochemistry, and Physiology of Human Steroidogenesis and Its Disorders (2011) · cites this paper as [240]

  • Computational modeling of P450c17 predicts the effects of all known mutations, including those with partial retention of both activities and those causing selective 17,20-lyase deficiency.

    “Computational modeling of P450c17 predicts the effects of all known mutations, including those with partial retention of both activities and those causing selective 17,20-lyase deficiency (240).”

    From The Molecular Biology, Biochemistry, and Physiology of Human Steroidogenesis and Its Disorders (2011) · cites this paper as [240]

  • Basic residues in the redox-partner-binding surface of the recipient P450 are crucial for electron transfer from POR, as shown by mutations in human P450c17 that cause 17,20-lyase deficiency.

    “Basic residues in the redox-partner-binding surface of the recipient P450 are crucial for electron transfer from POR (49, 302), as shown by the mutations in human P450c17 that cause 17,20lyase deficiency (240, 280).”

    From The Molecular Biology, Biochemistry, and Physiology of Human Steroidogenesis and Its Disorders (2011) · cites this paper as [240]

  • Genetic and biochemical studies have implicated basic residues in P450c17, including R347, R358, and perhaps R449 and K89, as important for its interaction with cytochrome b5, whereas E48 and E49 of cytochrome b5 are required for high 17,20-lyase activity.

    “Genetic and biochemical studies have implicated basic residues in P450c17, including R347, R358, and perhaps R449 and K89, as important for its interaction with cytochrome b 5 (240,280,348), whereas E48 and E49 of cytochrome b 5 are required for high 17,20-lyase activity (349).”

    From The Molecular Biology, Biochemistry, and Physiology of Human Steroidogenesis and Its Disorders (2011) · cites this paper as [240]

  • By analogy with the computationally inferred structure of P450c17, Arg 356 appears to be part of the redox-partner-binding site, Val 281 appears to participate in coordinating the heme moiety, and Cys 428 is the crucial cystine residue in the heme-binding site found in all cytochrome P450 enzymes.

    “By analogy with the computationally inferred structure of P450c17 (240), Arg 356 appears to be part of the redox-partner-binding site, Val 281 appears to participate in coordinating the heme moiety, and Cys 428 is the crucial cystine residue in the hemebinding site found in all cytochrome P450 enzymes.”

    From The Molecular Biology, Biochemistry, and Physiology of Human Steroidogenesis and Its Disorders (2011) · cites this paper as [240]

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Authors
Auchus, R. J., & Miller, W. L.
Affiliations
University of California, San Francisco (US)
Corresponding author
Miller, Walter L.
ORCID
Venue
Molecular Endocrinology
Publisher
The Endocrine Society
Published
1999-07
Volume
13
Issue
7
Pages
1169-1182
ISSN
  • 0888-8809 (ISSN-L)
  • 1944-9917
DOI
10.1210/mend.13.7.0326
OpenAlex
W2112100343
Cited by
185
References
71
Citations by year
2025: 3 · 2024: 1 · 2022: 2 · 2021: 4 · 2020: 1 · 2019: 1 · 2018: 1 · 2017: 2 · 2016: 5 · 2015: 7 · 2014: 8 · 2013: 8 · 2012: 8
FWCI
6.63
Type
journal article
Language
en
Open access
Locations
  • Molecular Endocrinology (publishedVersion)
  • PubMed (publishedVersion)
Funders
  • National Institutes of Health (P41 RP-01081, 5r01dk042154-09)
  • University of California, San Francisco
  • National Institute of Diabetes and Digestive and Kidney Diseases (DK-37922, DK 42154, R01DK037922)
  • Eunice Kennedy Shriver National Institute of Child Health and Human Development (U54-HD34449, U54HD034449)
Topics
  • Physical Sciences › Chemistry › Inorganic Chemistry › Metal-Catalyzed Oxygenation Mechanisms
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Structure and Dynamics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Steroid Chemistry and Biochemistry
Keywords
CYP17, 17α-hydroxylase/17,20-lyase, steroid hydroxylation, steroidogenesis, androgen production, estrogen synthesis, active site structure, substrate specificity, regioselectivity, hydrogen abstraction, pregnenolone, progesterone, molecular docking, molecular dynamics simulation
MeSH terms
Amino Acid Sequence, Bacterial Proteins, Binding Sites, Cytochrome P-450 Enzyme System, Heme, Humans, Mixed Function Oxygenases, Models, Molecular, Molecular Sequence Data, Mutation, NADPH-Ferrihemoprotein Reductase, Pregnenolone, Protein Conformation, Steroid 17-alpha-Hydroxylase, Mutagenesis, Site-Directed, Sequence Homology, Amino Acid

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