A comparative approach to structure-function studies of mammalian aromatases

Area of Science:

• Biochemistry
• Enzymology
• Molecular Biology

Background:

• Aromatase cytochrome P450 (P450arom) structure-function studies traditionally rely on human enzyme analyses and computational models.
• Recent identification of distinct porcine gonadal and placental P450arom isozymes suggests functional divergence.

Purpose of the Study:

• To delineate specific functional regions of P450arom using a comparative approach with porcine isozymes.
• To investigate the basis for differential inhibitor sensitivity between P450arom isozymes.

Main Methods:

• Expression of native and chimeric human and porcine P450arom constructs.
• Assessment of enzyme activity using the tritiated water assay.
• Investigation of inhibitor sensitivity (etomidate, CGS16949A) and protein expression via immunoblot analysis.

Main Results:

• All chimeric constructs retained catalytic activity, indicating structural element exchange does not abolish function.
• Porcine gonadal P450arom exhibited significantly higher sensitivity to etomidate inhibition compared to placental and human forms.
• Etomidate sensitivity was mapped to specific helices (B, B', C) in the porcine gonadal P450arom, distinct from substrate recognition sites.

Conclusions:

• A comparative approach using chimeric enzymes is effective for identifying functional regions in P450arom.
• Residues conferring etomidate sensitivity in porcine gonadal P450arom appear distinct from those involved in substrate binding and metabolism.
• These findings guide future point mutational analyses for understanding P450 inhibitor and substrate recognition.