Why human cytochrome P450c21 is a progesterone 21-hydroxylase

Dario Mizrachi, Zhu Wang, Kamalesh K. Sharma, Manisha K. Gupta, Keliang Xu, Christopher R. Dwyer, Richard J. Auchus

Research output: Contribution to journalArticle

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Abstract

Human cytochrome P450c21 (steroid 21-hydroxylase, CYP21A2) catalyzes the 21-hydroxylation of progesterone (P4) and its preferred substrate 17α-hydroxyprogestrone (17OHP4). CYP21A2 activities, which are required for cortisol and aldosterone biosynthesis, involve the formation of energetically disfavored primary carbon radicals. Therefore, we hypothesized that the binding of P4 and 17OHP4 to CYP21A2 restricts access of the reactive heme-oxygen complex to the C-21 hydrogen atoms, suppressing oxygenation at kinetically more favorable sites such as C-17 and C-16, which are both hydroxylated by cytochrome P450c17 (CYP17A1). We reasoned that expansion of the CYP21A2 substrate-binding pocket would increase substrate mobility and might yield additional hydroxylation activities. We built a computer model of CYP21A2 based principally on the crystal structure of CYP2C5, which also 21-hydroxylates P4. Molecular dynamics simulations indicate that binding of the steroid nucleus perpendicular to the plane of the CYP21A2 heme ring limits access of the heme oxygen to the C-21 hydrogen atoms. Residues L107, L109, V470, I471, and V359 were found to contribute to the CYP21A2 substate-binding pocket. Mutation of V470 and I471 to alanine or glycine preserved P4 21-hydroxylase activity, and mutations of L107 or L109 were inactive. Mutations V359A and V359G, in contrast, acquired 16α-hydroxylase activity, accounting for 40% and 90% of the P4 metabolites, respectively. We conclude that P4 binds to CYP21A2 in a fundamentally different orientation than to CYP17A1 and that expansion of the CYP21A2 substrate-binding pocket allows additional substrate trajectories and metabolic switching.

Original languageEnglish (US)
Pages (from-to)3968-3974
Number of pages7
JournalBiochemistry
Volume50
Issue number19
DOIs
StatePublished - May 17 2011

Fingerprint

Steroid 21-Hydroxylase
Heme
Hydroxylation
Mutation
Hydrogen
Substrates
Oxygen
Molecular Dynamics Simulation
Cytochromes
Mixed Function Oxygenases
Aldosterone
Alanine
Computer Simulation
Glycine
Progesterone
Hydrocortisone
Carbon
Atoms
Steroids
Oxygenation

ASJC Scopus subject areas

  • Biochemistry

Cite this

Mizrachi, D., Wang, Z., Sharma, K. K., Gupta, M. K., Xu, K., Dwyer, C. R., & Auchus, R. J. (2011). Why human cytochrome P450c21 is a progesterone 21-hydroxylase. Biochemistry, 50(19), 3968-3974. https://doi.org/10.1021/bi102078e

Why human cytochrome P450c21 is a progesterone 21-hydroxylase. / Mizrachi, Dario; Wang, Zhu; Sharma, Kamalesh K.; Gupta, Manisha K.; Xu, Keliang; Dwyer, Christopher R.; Auchus, Richard J.

In: Biochemistry, Vol. 50, No. 19, 17.05.2011, p. 3968-3974.

Research output: Contribution to journalArticle

Mizrachi, D, Wang, Z, Sharma, KK, Gupta, MK, Xu, K, Dwyer, CR & Auchus, RJ 2011, 'Why human cytochrome P450c21 is a progesterone 21-hydroxylase', Biochemistry, vol. 50, no. 19, pp. 3968-3974. https://doi.org/10.1021/bi102078e
Mizrachi D, Wang Z, Sharma KK, Gupta MK, Xu K, Dwyer CR et al. Why human cytochrome P450c21 is a progesterone 21-hydroxylase. Biochemistry. 2011 May 17;50(19):3968-3974. https://doi.org/10.1021/bi102078e
Mizrachi, Dario ; Wang, Zhu ; Sharma, Kamalesh K. ; Gupta, Manisha K. ; Xu, Keliang ; Dwyer, Christopher R. ; Auchus, Richard J. / Why human cytochrome P450c21 is a progesterone 21-hydroxylase. In: Biochemistry. 2011 ; Vol. 50, No. 19. pp. 3968-3974.
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abstract = "Human cytochrome P450c21 (steroid 21-hydroxylase, CYP21A2) catalyzes the 21-hydroxylation of progesterone (P4) and its preferred substrate 17α-hydroxyprogestrone (17OHP4). CYP21A2 activities, which are required for cortisol and aldosterone biosynthesis, involve the formation of energetically disfavored primary carbon radicals. Therefore, we hypothesized that the binding of P4 and 17OHP4 to CYP21A2 restricts access of the reactive heme-oxygen complex to the C-21 hydrogen atoms, suppressing oxygenation at kinetically more favorable sites such as C-17 and C-16, which are both hydroxylated by cytochrome P450c17 (CYP17A1). We reasoned that expansion of the CYP21A2 substrate-binding pocket would increase substrate mobility and might yield additional hydroxylation activities. We built a computer model of CYP21A2 based principally on the crystal structure of CYP2C5, which also 21-hydroxylates P4. Molecular dynamics simulations indicate that binding of the steroid nucleus perpendicular to the plane of the CYP21A2 heme ring limits access of the heme oxygen to the C-21 hydrogen atoms. Residues L107, L109, V470, I471, and V359 were found to contribute to the CYP21A2 substate-binding pocket. Mutation of V470 and I471 to alanine or glycine preserved P4 21-hydroxylase activity, and mutations of L107 or L109 were inactive. Mutations V359A and V359G, in contrast, acquired 16α-hydroxylase activity, accounting for 40{\%} and 90{\%} of the P4 metabolites, respectively. We conclude that P4 binds to CYP21A2 in a fundamentally different orientation than to CYP17A1 and that expansion of the CYP21A2 substrate-binding pocket allows additional substrate trajectories and metabolic switching.",
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AU - Mizrachi, Dario

AU - Wang, Zhu

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AU - Dwyer, Christopher R.

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