Martin Lisandra L, Holien Jessica K, Mizrachi Dario, Corbin C Jo, Conley Alan J, Parker Michael W, Rodgers Raymond J
School of Chemistry, Monash University, Clayton 3800, Victoria, Australia.
ACRF Rational Drug Discovery Centre, St Vincent's Institute of Medical Research, Fitzroy 3065, Victoria, Australia.
J Steroid Biochem Mol Biol. 2015 Nov;154:294-301. doi: 10.1016/j.jsbmb.2015.09.006. Epub 2015 Sep 7.
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.
雌激素是一种必需的脊椎动物激素，由雄激素经多次羟基化合成，由细胞色素P450芳香化酶（P450arom或CYP19）催化。尽管它们很重要，但在脊椎动物和/或哺乳动物的P450arom酶的结构或功能方面，很少有比较研究。在这里，我们直接比较了人（h-）和猪性腺（p(g)-）P450arom，因为p(g)-P450arom的催化效率非常低，对底物（雄烯二酮）的亲和力高十倍（Km），周转数（Vmax）降低十倍。我们重组表达了这些蛋白质，并使用石英晶体微天平（QCM）以及电子供体蛋白细胞色素P450氧化还原酶（CPR）比较了它们在膜上的相互作用。QCM中的频率和耗散变化支持h-P450arom形成同二聚体，这与荧光共振能量转移（FRET）数据一致，但p(g)-P450arom并非如此。对h-P450arom的X射线晶体结构分析表明了一个可能的同二聚化位点，并发现某些关键的相互作用残基在pg-P450arom中不保守。分子动力学模拟为这些残基在同二聚化中的重要性提供了支持。在这里，我们提出h-P450arom较低的亲和力和较高的活性以及中间代谢产物释放减少是其形成同二聚体能力的结果。二聚化的功能意义为高效芳香化的需求提供了一个重要的机制步骤。
