Crystal structures of ligand complexes of P450eryF exhibiting homotropic cooperativity

摘要

Several mammalian cytochrome P450 (P450) isoforms demonstrate homotropic cooperativity with a number of substrates, including steroids and polycyclic aromatic hydrocarbons. To identify struc- tural factors contributing to steroid and polycyclic aromatic hydro- carbon binding to P450 enzymes and to determine the location of the allosteric site, we investigated interactions of the macrolide hydroxylase P4S0eryF from Saccharopotwora erythraea with an- drostenedione and 9-aminophenanthrene. Spectroscopic binding assays indicate that P4S0eryF binds androstenedione with an affinity of 36S pM and a Hill coefficient of 1.31 t 0.6 and coordinates with 9-aminophenanthrene with an affinity of 91 pM and a Hill coefficient of 1.38 t 0.2. Crystals of complexes of androstenedione and 9-aminophenanthrene with P4S0eryF were grown and diffracted to 2.1 A and 2.35 A, respectively. Electron density maps indicate that for both complexes two ligand mole- cules are simultaneously present in the active site. The P450eryF/ androstenedione model was refined to an r = 18.9/100. and the two androstenedione molecules have similar con formations. The prox- imal androstenedione is positioned such that the α-face of car- bon-6 is closest to the heme iron, and the second steroid molecule is positioned. S.5 A distal in the active site. The P450eryF/9- aminophenanthrene model was refined to an r = 19.7/100 with the proximal 9-aminophenanthrene coordinated with the heme iron through the 9-amino group and the second ligand positioned ≈6 A distal in the active site. These results establish that homotropic cooperativity in ligand binding can result from binding of two substrate molecules within the active site pocket without major con formational changes in the protein.