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

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.