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...

The catalytic site contains residues Tyr152 and Lys156. These two amino acids are strictly conserved in the short-chain dehydrogenase superfamily. Modeling studies with a cortisone molecule in the catalytic site suggest that the Tyr152, Lys156 and Ser139 side chains promote electrophilic attack on the (C20-O) carbonyl oxygen atom, thus enabling the carbon atom to accept a hydride from the reduced cofactor.

The x-ray structure of a short-chain dehydrogenase, the bacterial holo 3a,20/3-hydroxysteroid dehydrogenase (EC 1.1.1.53), is described at 2.6 A resolution. This enzyme is active as a tetramer and crystallizes with four identical subunits in the asymmetric unit. It has the a/( fold characteristic ofthe dinucleotide binding region. The fold of the rest of the subunit, the quarternary structure, and the nature of the cofactor-enzyme interactions are, however, significantly different from those observed in the long-chain dehydrogenases. The architecture of the postulated active site is consistent with the observed stereospecificity of the enzyme and the fact that the tetramer is the active form. There is only one cofactor and one substrate-binding site per subunit; the specificity for both 3a-and 2013-ends of the steroid results from the binding of the steroid in two orientations near the same cofactor at the same catalytic site. (1), which includes 11f,-hydroxysteroid (llP-HSD) (2), 7a-hydroxysteroid (3), and 15-hydroxyprostaglandin dehydrogenases (4) from mammals; glucose (5) and ribitol (6) dehydrogenases, as well as a putative nodulation factor (7) from bacteria; and an ADH (8) from insects. Enzymes belonging to this family have -250 amino acid residues, similar coenzyme specificity, and partial sequence homology. Although more than 40 crystal structures of =15 types of NAD(H)-and NADP(H)-linked dehydrogenase enzymes have been determined at medium-to-high resolution (9), to our knowledge no x-ray crystallographic study describing the three-dimensional structure of a dehydrogenase belonging to this short-chain class has been reported. This is only the third structure of an enzyme for which steroids are the substrate to be determined by x-ray diffraction techniques. A lowresolution structure of keto-steroid isomerase (10) and the refined structure of cholesterol oxidase (11) have been published.To account for the ability of 3a,20f3-HSD to transfer a hydride to either end of a steroid molecule, "one steroid-two cofactor sites" and "two steroid orientations-one cofactor site" models (12) have been proposed. When analyzed in conjunction with sequence homology studies, the threedimensional structured especially at the cofactor binding and the substrate binding regions, offers further insight concerning the significance of conserved residues and their possible roles in substrate specificity and overall enzyme function. MATERIALS AND METHODSThe crystals, grown in the presence of 4 mM NADH, belong to the space group P43212 having unit cell dimensions a = 106.2 A and c = 203.8 A and contain one full tetramer (106 kDa) in the asymmetric unit (13 .091], was collected on film from six crystals at the Cornell High Energy Synchrotron Source and processed by using Rossmann's program at Purdue University. The area detector data to 3 A and film data between 3 and 2.6 A were merged into a composite native data set. The Hg derivatives each had a single major binding site per subunit, whereas the Au reagent gave a "multip...

The monomer structure is the same in both the complexed and uncomplexed crystal forms. The dimers differ in the relative positions of the two monomers at the dimer interface. Of the 55 residues that are different in CE from those in C. rugosa lipase 1, 23 are located in the active site and at the dimer interface. The altered substrate specificity is a direct consequence of these substitutions.

Excess 17␤-estradiol (E 2 ), the most potent of human estrogens, is known to act as a stimulus for the growth of breast tumors. Human estrogenic 17␤-hydroxysteroid dehydrogenase type 1 (17␤-HSD1), which catalyzes the reduction of inactive estrone (E 1 ) to the active 17␤-estradiol in breast tissues, is a key enzyme responsible for elevated levels of E 2 in breast tumor tissues. We present here the structure of the ternary complex of 17␤-HSD1 with the cofactor NADP ؉ and 3-hydroxyestra-1,3,5,7-tetraen-17-one (equilin), an equine estrogen used in estrogen replacement therapy. The ternary complex has been crystallized with a homodimer, the active form of the enzyme, in the asymmetric unit. Structural and kinetic data presented here show that the 17␤-HSD1-catalyzed reduction of E 1 to E 2 in vitro is specifically inhibited by equilin. The crystal structure determined at 3.0-Å resolution reveals that the equilin molecule is bound at the active site in a mode similar to the binding of substrate. The orientation of the 17-keto group with respect to the nicotinamide ring of NADP ؉ and catalytic residues Tyr-155 and Ser-142 is different from that of E 2 in the 17␤-HSD1-E 2 complex. The ligand and substrate-entry loop densities are well defined in one subunit. The substrate-entry loop adopts a closed conformation in this subunit. The result demonstrates that binding of equilin at the active site of 17␤-HSD1 is the basis for inhibition of E 1 -to-E 2 reduction by this equine estrogen in vitro. One possible outcome of estrogen replacement therapy in vivo could be reduction of E 2 levels in breast tissues and hence the reduced risk of estrogen-dependent breast cancer.17␤-Hydroxysteroid dehydrogenases (17␤-HSDs) are a group of enzymes that are involved in interconversion of active and inactive forms of androgens and estrogens (1-7) by NAD(P)(H)-linked oxidoreductive transfer of a hydride to and from the 17-position of steroid molecules. Six distinct 17␤-HSD isozymes, numbered 1-6, have been identified and cloned (2-7). These isozymes differ in specificities for substrate and tissue and in the preferred direction of the reaction. In human breast tissues, the most active estrogen, 17␤-estradiol (E 2 ), is formed by reduction of the inactive estrogen, estrone (E 1 ), which is catalyzed by 17␤-HSD type 1 (17␤-HSD1). The estrogenic specificity of 17␤-HSD1 as well as its preference for the reduction reaction has been well established (8-10).17␤-HSD1 is expressed in steroidogenic tissues including estrogen target tissues such as normal and malignant endometrium and breast tissues (11-16). Because of its estrogenic specificity and preference for the E 1 -to-E 2 reduction reaction, the enzyme is considered to be primarily responsible for E 2 biosynthesis in gonads and in peripheral tissues. This enzyme has been proposed to be involved in maintaining high E 2 levels found in breast tumors of postmenopausal women (ref. 17, and references therein). A direct correlation between higher concentrations of E 2 and onset of breast canc...