Structural Basis for the Functional Roles of Critical Residues in Human Cytochrome P450 Aromatase

Abstract

Cytochrome P450 aromatase (CYP19A1) is the only enzyme known to catalyze the biosynthesis of estrogens from androgens. The crystal structure of human placental aromatase (pArom) has paved the way toward understanding the structure–function relationships of this remarkable enzyme. Using an amino terminus-truncated recombinant human aromatase (rArom) construct, we investigate the roles of key amino acids in the active site, at the intermolecular interface, inside the access channel, and at the lipid–protein boundary for their roles in enzyme function and higher-order organization. Replacing the active site residue D309 with an N yields an inactive enzyme, consistent with its proposed involvement in aromatization. Mutation of R192 at the lipid interface, pivotal to the proton relay network in the access channel, results in the loss of enzyme activity. In addition to the distal catalytic residues, we show that mutation of K440 and Y361 of the heme-proximal region critically interferes with substrate binding, enzyme activity, and heme stability. The D–E loop deletion mutant Del7 that disrupts the intermolecular interaction significantly reduces enzyme activity. However, the less drastic Del4 and point mutants E181A and E181K do not. Furthermore, native gel electrophoresis, size-exclusion chromatography, and analytical ultracentrifugation are used to show that mutations in the intermolecular interface alter the quaternary organization of the enzyme in solution. As a validation for interpretation of the mutational results in the context of the innate molecule, we determine the crystal structure of rArom to show that the active site, tertiary, and quaternary structures are identical to those of pArom.

Effect of sildenafil on human aromatase activity: From in vitro structural analysis to catalysis and inhibition in cells

“…The activity of rArom (55 nmol/min/mg protein) [17] was previously demonstrated to be comparable to the full-length placental and crystallized enzyme [18] as well as other recombinant systems previously developed for the same enzyme [41][42][43][44][45].…”

“…This behaviour was assigned to a specific residue (Asp309) by site directed mutagenesis experiments [36]. According to the crystal structure, this residue is involved in a hydrogen bond with the keto-group of the substrate and it is protonated at physiological pH [17,18,36]. For the aromatase inhibitor anastrozole, the K D values variation did not show a linear increasing trend in the pH range 6.5-9.0, indicating that the inhibitor binding does not directly depends on the protonation state of Asp309 in the pH range investigated.…”

“…The structure of sildenafil shows the presence of a pyrazole ring often involved in aromatase inhibitors, such as anastrozole (Scheme 1 B-C). This paper investigates this hypothesis in vitro at molecular level, using the purified N-terminally modified recombinant aromatase (rArom) that we have shown having the same 3D structure of the full-length wild-type human enzyme (RMSD 0.4 Å) [17,18]. The in vitro data are validated using cell models expressing the fulllength enzyme, including breast cancer cells.…”

Evidence for an Elevated Aspartate pK in the Active Site of Human Aromatase

“…Although inferences from crystallography suggest protonation of Asp 309 , and theory highlights the potential value of this proton for catalysis, additional experimental evidence is required to support an elevated pK a for Asp 309 in aromatase. In a previous work, using an N-terminally truncated form of human aromatase expressed in Escherichia coli (rArom-WT) 3 and sharing the same structural and functional features of the full-length wild-type enzyme, we found that the mutation of Asp 309 to a non-titratable asparagine residue abolishes aromatase catalytic activity (12). In the present work, we describe the pH dependence of aromatase ligand binding.…”

“…Theoretical calculations based on one crystal structure (11) and site-directed mutagenesis studies (12) supported the structural data. Furthermore, the crystal structures in complex with androstenedione revealed a close contact (2.7 Å) between the 3-keto oxygen atom of androstenedione and the ␦-O atom of Asp 309 (10,12). Similar distances between these atoms in the crystal structures with exemestane and other steroidal inhibitors have likewise been observed (13).…”