Structure-function relationships of human aromatase cytochrome P-450 using molecular modeling and site-directed mutagenesis
The conversion of androgens to estrogens is catalyzed by an enzyme complex named aromatase, which consists of a form of cytochrome P-450, aromatase cytochrome P-450 (cytochrome P-450AROM), and the flavoprotein, NADPH-cytochrome P-450 reductase. As a first step toward investigation of the structure-f...
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Published in | The Journal of biological chemistry Vol. 266; no. 18; pp. 11939 - 11946 |
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Main Authors | , , , , |
Format | Journal Article |
Language | English |
Published |
Bethesda, MD
American Society for Biochemistry and Molecular Biology
25.06.1991
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Subjects | |
Online Access | Get full text |
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Summary: | The conversion of androgens to estrogens is catalyzed by an enzyme complex named aromatase, which consists of a form of cytochrome
P-450, aromatase cytochrome P-450 (cytochrome P-450AROM), and the flavoprotein, NADPH-cytochrome P-450 reductase. As a first
step toward investigation of the structure-function relationships of cytochrome P-450AROM, we have used computer modeling
to align the amino acid sequence of cytochrome P-450AROM with that of cytochrome P-450CAM from Pseudomonas putida and thus
create a substrate pocket using the heme-binding region and the I-helix of cytochrome P-450CAM as the template. Site-directed
mutagenesis was then carried out at two sites: one at a region that aligns with the bend in the I-helix of cytochrome P-450CAM
and the other at a glutamate (Glu302) just N-terminal of this bend, which is predicted to be in close proximity to the C2-position
of the androstenedione substrate. To determine the importance of the former region, three mutants were constructed: A307G
(Ala307---Gly), P308V (Pro308---Val), and GAGV, which changed -Ile305-Ala306-Ala307-Pro308- to -Gly-Ala-Gly-Val- (the corresponding
sequence found in 17 alpha-hydroxylase cytochrome P-450). When these proteins were expressed in COS-1 cells, it was found
that the activity of P308V was approximately one-third that of the wild type. These observations are consistent with the concept
that Pro308 causes a bend in the I-helix of cytochrome P-450AROM, similar to that observed in cytochrome P-450CAM, which is
believed to be important in forming the substrate-binding pocket. The next set of mutants were designed to determine the importance
of Glu302 in catalysis. Four mutants were prepared in which Glu302 was changed either to Ala, Val, Gln, or Asp, and the activities
of the expressed proteins were examined. It was found that mutations in which the carboxylic acid was replaced were essentially
devoid of activity. On the other hand, changing Glu302 to Asp resulted in a two-thirds reduction in the apparent Vmax. These
results support the role of a carboxylic acid residue at position 302 in the catalytic activity of cytochrome P-450AROM. |
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Bibliography: | ObjectType-Article-2 SourceType-Scholarly Journals-1 ObjectType-Feature-1 content type line 23 |
ISSN: | 0021-9258 1083-351X |
DOI: | 10.1016/S0021-9258(18)99048-4 |