title: Evolutionary comparisons predict that dimerization of human cytochrome P450 aromatase increases its enzymatic activity and efficiency.
abstract: Estrogen is an essential vertebrate hormone synthesized from androgens involving multiple hydroxylations, catalyzed by cytochrome P450 aromatase (P450arom or CYP19) enzymes. Despite their importance, very few comparative studies have been conducted on vertebrate and/or mammalian P450arom enzymes, either structurally or functionally. Here we directly compared the human (h-) and porcine gonadal (p(g)-) P450arom, as p(g)-P450arom has very low catalytic efficiency, with a ten-fold higher affinity (Km) for a substrate (androstenedione) and ten-fold reduction in turnover (Vmax). We recombinantly expressed these proteins and compared their interactions on a membrane using a quartz crystal microbalance (QCM) and also with the electron donor protein cytochrome P450 oxidoreductase (CPR). Changes in frequency and dissipation in the QCM supported the h-P450arom forming a homodimer that agreed with the FRET data, but not p(g)-P450arom. Analysis of the X-ray crystal structure of the h-P450arom suggested a likely site of homo-dimerization and found that certain key interacting residues were not conserved in pg-P450arom. Molecular dynamics simulations provide support for the importance of these residues in homo-dimerization. Here we propose that the lower affinity and higher activity with reduced release of intermediate metabolites by the h-P450arom is as a consequence of its ability to form homodimers. The functional implications of dimerization provide an important mechanistic step in the requirement for efficient aromatization.
authors.affiliation: School of Chemistry, Monash University, Clayton 3800, Victoria, Australia. Electronic address: Lisa.Martin@monash.edu.
authors.forename: Lisandra L
authors.initials: LL
authors.lastname: Martin
authors.affiliation: ACRF Rational Drug Discovery Centre, St Vincent's Institute of Medical Research, Fitzroy 3065, Victoria, Australia.
authors.forename: Jessica K
authors.initials: JK
authors.lastname: Holien
authors.affiliation: Chemical and Biomolecular Engineering, College of Engineering, Cornell University, Ithaca, NY, USA.
authors.forename: Dario
authors.initials: D
authors.lastname: Mizrachi
authors.affiliation: School of Veterinary Medicine, University of California, Davis, USA.
authors.forename: C Jo
authors.initials: CJ
authors.lastname: Corbin
authors.affiliation: School of Veterinary Medicine, University of California, Davis, USA.
authors.forename: Alan J
authors.initials: AJ
authors.lastname: Conley
authors.affiliation: ACRF Rational Drug Discovery Centre, St Vincent's Institute of Medical Research, Fitzroy 3065, Victoria, Australia; Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Parkville 3010, Victoria, Australia.
authors.forename: Michael W
authors.initials: MW
authors.lastname: Parker
authors.affiliation: Discipline of Obstetrics and Gynaecology, School of Medicine, Robinson Research Institute, The University of Adelaide, SA 5005, Australia.
authors.forename: Raymond J
authors.initials: RJ
authors.lastname: Rodgers
mesh_terms: D000818:Animals; D001141:Aromatase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D019143:Evolution, Molecular; D006801:Humans; D056004:Molecular Dynamics Simulation; D058525:Quartz Crystal Microbalance Techniques; D013552:Swine
chemical_list: D001141:Aromatase
keywords: Cytochrome P450 aromatase; Dimerization; Estrogen; Molecular dynamics; protein–protein interaction