Amino acid substitutions Phe66 → Leu and Ser126 → Pro abolish cortisol and aldosterone synthesis by bovine cytochrome P450(11β)

P. A. Mathew, J. I. Mason, J. M. Trant, D. Sanders, M. R. Waterman

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Abstract

A cDNA clone encoding the complete protein sequence of the precursor form of bovine cytochrome P450(11β) has been constructed using a combined technique of first strand cDNA synthesis by reverse transcription followed by polymerase chain reaction. Upon expression of this cDNA in COS 1 cells the P450(11β) is found to be proteolytically processed and localized in the mitochondrion. This cDNA encodes the major form of P450(11β) found in bovine adrenal cortex (designated 11β-3; Kirita, S., Morohashi, K., Hashimoto, T., Yoshioka, H., Fujii-Kuriyama, Y., and Omura, T. (1988) J. Biochem. 104, 683-686) and is capable of catalyzing 11β-hydroxylation of deoxycorticosterone, 11-deoxycortisol, and androstenedione in COS 1 cells as well as aldosterone synthesis from deoxycorticosterone. In addition, a second form of P450(11β) (herein designated 11β-4), having no detectable 11β-hydroxylase activity or aldesterone synthase activity, was found in the local bovine population by this cloning procedure. These two forms of P450(11β) (11β-3 and 11β-4) contain five amino acid differences between them, all located within the amino-terminal half of the molecules. By changing 2 of the amino acids in the inactive form to the corresponding amino acids in the active form (Leu66 → Phe and Pro126 → Ser) both 11β-hydroxylase and aldosterone synthetase activities were completely restored. Neither of these changes alone led to detectable activity. Thus, upon expression in mitochondria of heterologous cells, bovine P450(11β) catalyzes both 11β-hydroxylation and aldosterone synthesis as reported previously for the purified enzyme in an in vitro reconstituted system, and Phe66 and Ser126 seem to be important residues in maintaining both activities.

Original languageEnglish
Pages (from-to)20228-20233
Number of pages6
JournalJournal of Biological Chemistry
Volume265
Issue number33
StatePublished - 18 Dec 1990

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