Steroid 17a-hydroxylase and 17, 20-lyase activities of p450cl7: Contributions of serine106 and p450 reductase

Dong Lin, Stephen Matthew Black, Yoshitaka Nagahama, Walter L. Miller

Research output: Contribution to journalArticle

172 Citations (Scopus)

Abstract

Cytochrome P450cl7 (EC 1.14.99.9) catalyzes both 17«-hydroxylase and 17, 20-lyase activities in mammalian steroidogenesis and also has some 16a-hydroxylase activity. The ratio of 17a-hydroxylase to 17, 20- lyase activity differs in the adrenal and testis and is developmentally regulated at adrenarche, but the nature of the enzyme’s active site and the differential regulation of its two principal activities are unknown. The spontaneous human P450cl7 mutation Ser10fi—»Pro eliminates all enzymatic activity. We used site-directed mutagenesis to construct expression vectors for the conservative P450cl7 mutations Ser1*—»Thr and Ser106—>Ala. When expressed in transfected COS-1 cells, these mutants retain only 20-30% of the 17a-hydroxylase and 17, 20-lyase activities, but retain 60% of the 16a-hydroxylase activity of the Ser101’ wild type. Thus, the amino acid occupying postion 106 greatly affects enzymatic activity. Ser is found at position 106 in P450cl7 in all mammals and birds studied, but the corresponding residue (position 112) in fish (trout) is Thr. Both the trout Thr112 wild type and a Thr112—>Ser trout mutant had equivalent 16«-hydroxylase, 170-hydroxylase, and 17, 20-lyase activities, although these were only 5%, 5%, and 10%, respectively, of human Ser106. To catalyze its activities, P450cl7 must receive electrons from NADPH via a flavoprotein termed P450 reductase. We examined the influence of the ratio of P450cl7 to P450 reductase on enzymatic activity by cotransfecting COS-1 cells with varying amounts of vectors expressing each protein. The endogenous P450 reductase of COS-1 cells was sufficient to confer maximal 17a-hydroxylase activity. P450 reductase produced from the transfected expression vector did not increase the conversion of [, 4C]progesterone to 17«- or 16a-hydroxy- progesterone, indicating that the endogenous immunodetectable P450 reductase of COS-1 cells was sufficient to confer maximal 17«-hydrox- ylase activity. By contrast, the additional P450 reductase produced by the expression vector increased 17, 20-lyase activity about 3-fold. Thus, the availability of reducing equivalents is a crucial factor in regulating 17, 20-lyase activity. P450 reductase also increased the 17, 20-lyase activity of the Thr1"1’ and Ala1* mutants. These data suggest that the essential role of Ser1* is in the active site, rather than in interacting with P450 reductase, and that electron transfer may play an important role in regulating the 17, 20-lyase activity of P450cl7.

Original languageEnglish (US)
Pages (from-to)2498-2506
Number of pages9
JournalEndocrinology
Volume132
Issue number6
DOIs
StatePublished - Jan 1 1993

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Steroid Hydroxylases
Steroid 17-alpha-Hydroxylase
Mixed Function Oxygenases
Oxidoreductases
COS Cells
Trout
Progesterone
Catalytic Domain
Adrenarche
Electrons
Flavoproteins
Mutation
Cytochromes
Site-Directed Mutagenesis
NADP
Birds
Testis
Mammals
Fishes

ASJC Scopus subject areas

  • Endocrinology

Cite this

Steroid 17a-hydroxylase and 17, 20-lyase activities of p450cl7 : Contributions of serine106 and p450 reductase. / Lin, Dong; Black, Stephen Matthew; Nagahama, Yoshitaka; Miller, Walter L.

In: Endocrinology, Vol. 132, No. 6, 01.01.1993, p. 2498-2506.

Research output: Contribution to journalArticle

Lin, Dong ; Black, Stephen Matthew ; Nagahama, Yoshitaka ; Miller, Walter L. / Steroid 17a-hydroxylase and 17, 20-lyase activities of p450cl7 : Contributions of serine106 and p450 reductase. In: Endocrinology. 1993 ; Vol. 132, No. 6. pp. 2498-2506.
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abstract = "Cytochrome P450cl7 (EC 1.14.99.9) catalyzes both 17«-hydroxylase and 17, 20-lyase activities in mammalian steroidogenesis and also has some 16a-hydroxylase activity. The ratio of 17a-hydroxylase to 17, 20- lyase activity differs in the adrenal and testis and is developmentally regulated at adrenarche, but the nature of the enzyme’s active site and the differential regulation of its two principal activities are unknown. The spontaneous human P450cl7 mutation Ser10fi—»Pro eliminates all enzymatic activity. We used site-directed mutagenesis to construct expression vectors for the conservative P450cl7 mutations Ser1*—»Thr and Ser106—>Ala. When expressed in transfected COS-1 cells, these mutants retain only 20-30{\%} of the 17a-hydroxylase and 17, 20-lyase activities, but retain 60{\%} of the 16a-hydroxylase activity of the Ser101’ wild type. Thus, the amino acid occupying postion 106 greatly affects enzymatic activity. Ser is found at position 106 in P450cl7 in all mammals and birds studied, but the corresponding residue (position 112) in fish (trout) is Thr. Both the trout Thr112 wild type and a Thr112—>Ser trout mutant had equivalent 16«-hydroxylase, 170-hydroxylase, and 17, 20-lyase activities, although these were only 5{\%}, 5{\%}, and 10{\%}, respectively, of human Ser106. To catalyze its activities, P450cl7 must receive electrons from NADPH via a flavoprotein termed P450 reductase. We examined the influence of the ratio of P450cl7 to P450 reductase on enzymatic activity by cotransfecting COS-1 cells with varying amounts of vectors expressing each protein. The endogenous P450 reductase of COS-1 cells was sufficient to confer maximal 17a-hydroxylase activity. P450 reductase produced from the transfected expression vector did not increase the conversion of [, 4C]progesterone to 17«- or 16a-hydroxy- progesterone, indicating that the endogenous immunodetectable P450 reductase of COS-1 cells was sufficient to confer maximal 17«-hydrox- ylase activity. By contrast, the additional P450 reductase produced by the expression vector increased 17, 20-lyase activity about 3-fold. Thus, the availability of reducing equivalents is a crucial factor in regulating 17, 20-lyase activity. P450 reductase also increased the 17, 20-lyase activity of the Thr1{"}1’ and Ala1* mutants. These data suggest that the essential role of Ser1* is in the active site, rather than in interacting with P450 reductase, and that electron transfer may play an important role in regulating the 17, 20-lyase activity of P450cl7.",
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