Structural basis for androgen specificity and oestrogen synthesis in human aromatase

Abstract

Aromatase cytochrome P450 is the only enzyme in vertebrates known to catalyse the biosynthesis of all oestrogens from androgens [1][2][3] . Aromatase inhibitors therefore constitute a front-line therapy for oestrogen-dependent breast cancer 3,4 . 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 16α-hydroxytestosterone to oestrone, 17β-oestradiol and 17β,16α-oestriol, respectively [1][2][3] . 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, fulllength 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 nextgeneration aromatase inhibitors.Human aromatase is the product of the CYP19A1 gene on chromosome 15q21.1 and consists of a haem group and a polypeptide chain of 503 amino-acid residues. Although aromatase has been extensively studied for more than 35 years [1][2][3][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19], the mechanism of the aromatization step remains poorly understood. Many soluble bacterial P450s, such as P450cam20 and P450eryF21, as well as recombinant human microsomal P450s, such as 3A4 (ref. 22), 2D6 (ref. 23) and 2A6 (ref. 24), that metabolize drug/xenobiotics, have beenCorrespondence and requests for materials should be addressed to D.G. (ghosh@hwi.buffalo.edu). Full Methods and any associated references are available in the online version of the paper at www.nature.com/nature.Supplementary Information is linked to the online version of the paper at www.nature.com/nature. Author Contributions J.G. and M.E. performed the purification and crystallization of aromatase. W.P. and J.G. contributed to diffraction data collection. D.G. was involved in diffraction data collection and processing. D.G. solved the structure, wrote the manuscript and was responsible for overall planning and supervision of the proje...

Architecture of a single membrane spanning cytochrome P450 suggests constraints that orient the catalytic domain relative to a bilayer

“…This finding was supported by atomic force microscopy, which showed the catalytic domain protrudes only 3.5 ± 1 nm above the membrane (35). A crystal structure of full-length aromatase at 2.9-Å resolution has been modeled, but weak electron density meant that the structures of 44 N-terminal and seven C-terminal residues could not be determined (36). Models that place the mouth of the substrate channel in direct contact with the hydrophobic environment of the lipid bilayer required the catalytic domain helices A′ and A to be imbedded and partially imbedded, respectively, in the lipid bilayer (28,37).…”

Discovery of novel aromatase inhibitors using a homogeneous time-resolved fluorescence assay

“…The crystal structure of the enzyme was obtained from the Protein Data Bank (PDB). According to previous literature [39] , the resi- Figure 8A), which may explain its affinity for the enzyme. Furthermore, one of the benzonitrile groups extended into the access channel that links the active site to the outer surface.…”

Egonol gentiobioside and egonol gentiotrioside from Styrax perkinsiae promote the biosynthesis of estrogen by aromatase

“…Recently, the crystal structure of aromatase derived from human placenta was successfully elucidated and shed some light on this process (Ghosh et al, 2009). However, the proposed androgen-aromatase interaction mechanism was static, and it is still unknown whether any conformational changes occur during estrogen biosynthesis.…”