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Engineering a mineralocorticoid to a glucocorticoid synthesizing cytochrome P450

Item Type:Article
Title:Engineering a mineralocorticoid to a glucocorticoid synthesizing cytochrome P450
Creators Name:Boettner, B. and Schrauber, H. and Bernhardt, R.
Abstract: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.
Keywords:Aldosterone Synthase, Amino Acid Sequence, Amino Acid Sequence Homology, Base Sequence, Cytochrome P-450 Enzyme System, DNA Primers, Molecular Models, Molecular Sequence Data, Protein Engineering, Recombinant Proteins, Secondary Protein Structure, Sequence Alignment, Site-Directed Mutagenesis, Steroid 11-beta-Hydroxylase, Structure-Activity Relationship, Tertiary Protein Structure
Source:Journal of Biological Chemistry
Publisher:American Society for Biochemistry and Molecular Biology
Page Range:8028-8033
Date:5 April 1996
Official Publication:https://doi.org/10.1074/jbc.271.14.8028
PubMed:View item in PubMed

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