Structure-function relationships of human aromatase cytochrome P-450 using molecular modeling and site-directed mutagenesis

Sandra Graham-Lorence, M. Wahid Khalil, Matthew C. Lorence, Carole R. Mendelson, Evan R. Simpson

Research output: Contribution to journalArticle

102 Scopus citations

Abstract

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-450(AROM)), and the flavoprotein, NADPH-cytochrome P-450 reductase. As a first step toward investigation of the structure-function relationships of cytochrome P-450(AROM), we have used computer modeling to align the amino acid sequence of cytochrome P-450(AROM) with that of cytochrome P-450(CAM) from Pseudomonas putida and thus create a substrate pocket using the heme-binding region and the I-helix of cytochrome P-450(CAM) 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-450(CAM) and the other at a glutamate (Glu302) just N-terminal of this bend, which is predicted to be 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 α-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-450(AROM), similar to that observed in cytochrome P-450(CAM), 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 V(max). These results support the role of a carboxylic acid residue at position 302 in the catalytic activity of cytochrome P-450(AROM).

Original languageEnglish (US)
Pages (from-to)11939-11946
Number of pages8
JournalJournal of Biological Chemistry
Volume266
Issue number18
StatePublished - 1991

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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