A three‐dimensional model of aromatase cytochrome P450

Abstract

P450 hemeproteins comprise a large gene superfamily that catalyzes monooxygenase reactions in the presence of a redox partner. Because the mammalian members are, without exception, membrane-bound proteins, they have resisted structure-function analysis by means of X-ray crystallographic methods. Among P450-catalyzed reactions, the aromatase reaction that catalyzes the conversion of C19 steroids to estrogens is one of the most complex and least understood. Thus, to better understand the reaction mechanism, we have constructed a three-dimensional model of P450arom not only to examine the active site and those residues potentially involved in catalysis, but to study other important structural features such as substrate recognition and redox-partner binding, which require examination of the entire molecule (excepting the putative membrane-spanning region). This model of P450arom was built based on a "core structure" identified from the structures of the soluble, bacterial P450s (P450cam, P450terp, and P450BM-P) rather than by molecular replacement, after which the less conserved elements and loops were added in a rational fashion. Minimization and dynamic simulations were used to optimize the model and the reasonableness of the structure was evaluated. From this model we have postulated a membrane-associated hydrophobic region of aliphatic and aromatic residues involved in substrate recognition, a redoxpartner binding region that may be unique compared to other P450s, as well as residues involved in active site orientation of substrates and an inhibitor of P450arom, namely vorozole. We also have proposed a scheme for the reaction mechanism in which a "threonine switch" determines whether oxygen insertion into the substrate molecule involves an oxygen radical or a peroxide intermediate.

Membrane-Protein Interactions Contribute to Efficient 27-Hydroxylation of Cholesterol by Mitochondrial Cytochrome P450 27A1

“…The putative F-G loop is predicted to contribute to membrane association in microsomal P450s (10,11) and shown to contain residues involved in the interaction with the lipid bilayer in microsomal P450s 2B1, 2C5, and 7A1 (12)(13)(14). The present work is the first study showing that the putative F-G loop and the N-terminal part of the putative G helix are also the sites of membrane attachment in mitochondrial P450 27A1.…”

“…Another possible common site of association with the endoplasmic reticulum is an area between the F and G helices, the F-G loop (10 -14). The F-G loop was also proposed to form a mouth of the channel that allows the substrate to pass from the surface of the P450 molecule to the buried active site (10,11). Our recent studies indicate that membrane binding and substrate access merge in cholesterol-metabolizing enzyme, microsomal P450 7A1, and the putative F-G loop is simultaneously the site of attachment to membrane and cholesterol entry in this P450 (14).…”

Raman Evidence for Specific Substrate-Induced Structural Changes in the Heme Pocket of Human Cytochrome P450 Aromatase during the Three Consecutive Oxygen Activation Steps

“…In the Thr switch model, 19-aldo-AD would induce the displacement of distal Thr-310 through the hydrogen-bonding interaction for the formation of ferric peroxo species instead of compound I (24). On the basis of the Thr switch model and our current results, we propose the heme active site structure of the oxygen complex of P450arom as shown in Figure 8.…”

“…This observation raises the possibility that the orientation of the C19 group in 19-aldo-AD-bound P450arom would be different from those of other substrate-bound P450aroms and would not block the CO binding. Graham-Lorence et al proposed that the conserved Thr (Thr-310) at the heme distal site could be hydrogen-bonded to the oxygen atom of the aldehyde group in 19-aldo-AD to enhance the positive character on the carbonyl carbon and promote the nucleophilic attack of the ferric peroxo species in the third oxidative step (24). It is, therefore, likely that the hydrogen-bonding interaction between 19-aldo-AD and Thr-310 could induce the displacement of the C19 group in the 19-aldo-AD-bound P450arom to reduce the steric hindrance for CO binding (Figure 7d (Table 3), indicating that the FeCO unit adopts a bent conformation ( Figure 7d).…”

Memetic algorithms for ligand expulsion from protein cavities

“…Cytochrome P450 enzymes are heme-containing protein monooxygenases that are related to the synthesis and degradation of many physiologically crucial compounds and in the degradation of xenobiotics 15 . We studied bacterial cytochrome P450cam from Pseudomonas putida docked with heme and its natural substrate -camphor (PDB ID: 2CPP).…”