Structural basis for androgen specificity and oestrogen synthesis in human aromatase - PubMed (original) (raw)

Structural basis for androgen specificity and oestrogen synthesis in human aromatase

Debashis Ghosh et al. Nature. 2009.

Abstract

Aromatase cytochrome P450 is the only enzyme in vertebrates known to catalyse the biosynthesis of all oestrogens from androgens. Aromatase inhibitors therefore constitute a frontline therapy for oestrogen-dependent breast cancer. In a three-step process, each step requiring 1 mol of O(2), 1 mol of NADPH, and coupling with its redox partner cytochrome P450 reductase, aromatase converts androstenedione, testosterone and 16alpha-hydroxytestosterone to oestrone, 17beta-oestradiol and 17beta,16alpha-oestriol, respectively. The first two steps are C19-methyl hydroxylation steps, and the third involves the aromatization of the steroid A-ring, unique to aromatase. Whereas most P450s are not highly substrate selective, it is the hallmark androgenic specificity that sets aromatase apart. The structure of this enzyme of the endoplasmic reticulum membrane has remained unknown for decades, hindering elucidation of the biochemical mechanism. Here we present the crystal structure of human placental aromatase, the only natural mammalian, full-length P450 and P450 in hormone biosynthetic pathways to be crystallized so far. Unlike the active sites of many microsomal P450s that metabolize drugs and xenobiotics, aromatase has an androgen-specific cleft that binds the androstenedione molecule snugly. Hydrophobic and polar residues exquisitely complement the steroid backbone. The locations of catalytically important residues shed light on the reaction mechanism. The relative juxtaposition of the hydrophobic amino-terminal region and the opening to the catalytic cleft shows why membrane anchoring is necessary for the lipophilic substrates to gain access to the active site. The molecular basis for the enzyme's androgenic specificity and unique catalytic mechanism can be used for developing next-generation aromatase inhibitors.

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Figures

Figure 1

Figure 1. The structure of aromatase

a, A ribbon diagram showing the overall structure. The N terminus, starting at residue 45, is coloured dark blue and the C terminus ending at residue 496 is coloured red. The α-helices are labelled from A to L and β-strands are numbered from 1 to 10. The haem group, the bound androstenedione molecule at the active site and its polar interactions are shown. b, A close-up view of the active site showing the bound androstenedione molecule in unbiased difference (_F_obs − _F_cal) electron density contoured at 4.5 times the standard deviation. c, Modelling of Fe(III) as an oxyferryl Fe(IV)=O moiety. The C19-methyl hydrogen atoms are shown at the calculated ideal positions. Important side chains, haem and water molecules are depicted in element colours: grey, C; blue, N; red, O; yellow, S; firebrick, Fe; orange, H. The C atoms of androstenedione are coloured cornflower blue. This colour code is used in all figures. Distances are in ångströms. The directions of view into the active site are roughly similar in all panels. Unless otherwise noted, all three-dimensional illustrations were prepared with Chimera.

Figure 2

Figure 2. Views of the active site of aromatase

a, A van der Waals interaction surface cast by the protein and haem atoms at the active site. The semi-transparent surface, coloured green for hydrophobic interactions and magenta for polar interactions, closely resembles the shape, size and puckering of the steroid backbone. This figure was prepared with MOE. b, A view along the I-helix axis from its N-terminal end. The disruption to the helicity of the backbone at residues Pro 308-Asp 309-Thr 310 causes the helix axis to displace by about 3.5 Å, allowing the side chain of Asp 309 to interact with the 3-keto oxygen of the steroid. The deviation from helicity could be stabilized by a strong Ala 306CO···HOThr 310 (2.8 Å) hydrogen bond, as well as by Asp 309 peptide CO···water (3.4 Å) interaction as indicated.

Figure 3

Figure 3. Steroid–protein interactions and mechanistic implications

a, A close-up view of the Ala 306CO···HOThr 310 pair that may function in the aromatization of the A-ring of androstenedione. Calculated hydrogen-atom positions of C2 of the bound androstenedione are shown. Distances are in ångströms. b, A possible mechanism for H2β abstraction and 2,3-enolization that could be initiated by a nucleophilic attack on C2-H2β by the Ala 306CO···HOThr 310 pair, along with an electrophilic attack on the C3 carbonyl by a protonated Asp 309 side chain. The direction of proton flow from the proton relay network through Asp 309 carboxylate to the substrate is indicated by arrows. Involvement of a catalytic water in H2β abstraction is a possibility. The backbone carbonyl of the Ala 306CO···HOThr 310 pair aided by a potential catalytic water molecule, or the water oxygen itself (as indicated by dotted arrow) could act as the nucleophile. H1β abstraction is drawn as proposed previously. c, Modelling of an exemestane molecule (C atoms in magenta) after the experimental positioning of androstenedione. The short van der Waals contact distance (3 Å) between the C6-methylidene carbon and Cγ of Thr 310 is indicated by a black line.

Figure 4

Figure 4. A putative active-site access channel from within the lipid bilayer

a, The solvent-excluded surface of aromatase excludes the steroid-binding pocket and haem from the protein interior by forming a ‘pouch’-like cleft that has the only opening to the protein exterior through a channel, roughly at the arrowhead. The course of the polypeptide chain is shown in rainbow colour. Residues Arg 192, Asp 309, Ser 478 and Glu 483 border this channel from the protein interior; three water molecules, part of the proton relay network, are within the channel. The inset is a view along this channel at the arrowhead, showing the locations of water molecules and opening to the active site. b, In a proposed membrane integration model, the opening to the active-site access channel rests on the lipid bilayer surface, allowing the steroids to enter the aromatase active site directly from within the bilayer, roughly along the arrow shown. The model suggests lipid integration/association of the N terminus up to helix A, and other loops near the C terminus. The orientation of aromatase is roughly the same in both panels.

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