Engineering a mineralocorticoid- to a glucocorticoid-synthesizing cytochrome P450.
Site-directed mutagenesis of a domain (amino acids 299-338) aligning to the I-helix region of P450cam, P450BM3 and P450terp was used to investigate the different regioselectivities displayed in the hydroxylation reactions performed by human aldosterone synthase (P450aldo) and 11beta-hydroxylase (P45011beta). The two enzymes are 93% identical and are essential for the synthesis of mineralocorticoids and glucocorticoids in the human adrenal gland. Single replacement of P450aldo residues for P45011 beta-specific residues at positions 296, 301, 302, 320, and 335 only gave rise to slightly increased 11beta-hydroxylase activities. However, a L301P/A320V double substitution increased 11beta-hydroxylase activity to 60% as compared with that of P45011 beta. Additionally substituting Ala-320 for Val-320 of P45011 beta further enhanced this activity to 85%. The aldosterone synthase activities of the mutant P450aldo proteins were suppressed to a varying degree, with triple replacement mutant L301P/E302D/A320V retaining only 10% and double replacement mutant L301P/A320V retaining only 13% of the P450aldo wild type activity. These results demonstrate a switch in regio- and stereoselectivities of the engineered P450aldo enzyme due to manipulation of residues at three critical positions, and we attribute the determination of these features in P450aldo to the structure of a region analogous to the I-helix in P450cam.
Böttner
B
B
Max-Delbrueck-Centrum für Molekulare Medizin, Robert-Rössle-Strasse-10, D-13122 Berlin, Germany.
Schrauber
H
H
Bernhardt
R
R
D000595:Amino Acid Sequence; D001483:Base Sequence; D019405:Cytochrome P-450 CYP11B2; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry*; D017931:DNA Primers / Q000737:chemistry; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015202:Protein Engineering; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013252:Steroid 11-beta-Hydroxylase / Q000737:chemistry*; D013329:Structure-Activity Relationship
D017931:DNA Primers; D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System; D019405:Cytochrome P-450 CYP11B2; D013252:Steroid 11-beta-Hydroxylase