Structure-function studies of human aromatase

Chen, Shiuan; Zhou, Dujin; Swiderek, Kristine M.; Kadohama, N.; Osawa, Yoshio; Hall, Peter F.

doi:10.1016/0960-0760(93)90238-r

Cited by 40 publications

(15 citation statements)

References 26 publications

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“…However, because asparagine has no dissociable proton, it would not be able to participate in enzyme catalysis. 1 The tritiated water assay confirms that the D309N mutant is virtually inactive, 13,30 in agreement with the proposed mechanism. 1 This result shows that D309 is indeed essential for enzyme catalysis, being in the path of the proton relay network for the 3-keto enolization reaction.…”

Section: Discussionsupporting

confidence: 83%

“…However, without a recombinant, active enzyme that can be manipulated by site-directed mutagenesis, it is not possible to fully explore the molecular basis for catalysis, higher-order organization, and coupling to cytochrome P450 reductase (CPR). Although many laboratories to date recombinantly expressed and purified modified human Arom from various sources, 5–13 none yielded them in sufficient quality and/or quantity to be crystallized. Furthermore, mutagenesis studies in the past were mostly conducted in whole cells, 11,12,14,15 and a few in the purified enzyme.…”

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confidence: 99%

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

Nardo

Griswold

et al. 2013

Biochemistry

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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.

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Section: Discussionsupporting

confidence: 83%

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confidence: 99%

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

Nardo

Griswold

et al. 2013

Biochemistry

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“…Nevertheless, the enzymatic mechanism of estrogen formation and the role of these acidic residues is not totally understood. Earlier site directed mutagenesis studies on D309 of human aromatase implied the probable role of this aspartate in decarboxylation of androstenedione or testosterone and attracting a proton at C2 of the steroids during aromatization [7][8][9]. This process is assumed to be assisted by basic residues H475 and K473 present in proximity to C3 [10].…”

Section: Introductionmentioning

confidence: 99%

Active site acidic residues and structural analysis of modelled human aromatase: A potential drug target for breast cancer

Murthy

Nagaraju

Sastry

et al. 2006

J Comput Aided Mol Des

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This study sheds new light on the role of acidic residues present in the active site cavity of human aromatase. Eight acidic residues (E129, D222, E245, E302, D309, E379, D380 and D476) lining the cavity are identified and studied using comparative modeling, docking, molecular dynamics as well as statistical techniques. The structural environment of these acidic residues is studied to assess the stability of the corresponding carboxylate anions. Results indicate that the environment of the residues E245, E302 and D222 is most suitable for carboxylate ion formation in the uncomplexed form. However, the stability of D309, D222 and D476 anions is seen to increase on complexation to steroidal substrates. In particular, the interaction between D309 and T310, which assists proton transfer, is found to be formed following androgen/nor-androgen complexation. The residue D309 is found to be clamped in the presence of substrate which is not observed in the case of the other residues although they exhibit changes in properties following substrate binding. Information entropic analysis indicates that the residues D309, D222 and D476 have more conformational flexibility compared to E302 and E245 prior to substrate binding. Interaction similar to that between D476 and D309, which is expected to assist androgen aromatization, is proposed between E302 and E245. The inhibition of aromatase activity by 4-hydroxy androstenedione (formestane) is attributed to a critical hydrogen bond formation between the hydroxy moiety and T310/D309 as well as the large distance from D476. The results corroborate well with earlier site directed mutagenesis studies.

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“…Some of the residues derived by homology modeling and implicated in these proposed mechanisms are, in fact, present in the active site. For instance, several studies proposed Asp309 to be a catalytically important residue [2,23,28–30,33]. However, unlike the proposed models, Asp309 appears to be involved not only in catalysis but also in substrate binding.…”

Section: Resultsmentioning

confidence: 99%

X-ray structure of human aromatase reveals an androgen-specific active site

Ghosh

Griswold

Erman

et al. 2010

The Journal of Steroid Biochemistry and Molecular Biology

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Aromatase is a unique cytochrome P450 that catalyzes the removal of the 19-methyl group and aromatization of the A-ring of androgens for the synthesis of estrogens. All human estrogens are synthesized via this enzymatic aromatization pathway. Aromatase inhibitors thus constitute a frontline therapy for estrogen-dependent breast cancer. Despite decades of intense investigation, this enzyme of the endoplasmic reticulum membrane has eluded all structure determination efforts. We have determined the crystal structure of the highly active aromatase purified from human placenta, in complex with its natural substrate androstenedione. The structure shows the binding mode of androstenedione in the catalytically active oxidized high-spin ferric state of the enzyme. Hydrogen bond forming interactions and tight packing hydrophobic side chains that complement the puckering of the steroid backbone provide the molecular basis for the exclusive androgenic specificity of aromatase. Locations of catalytic residues and water molecules shed new light on the mechanism of the aromatization step. The structure also suggests a membrane integration model indicative of the passage of steroids through the lipid bilayer.

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Structure-function studies of human aromatase

Cited by 40 publications

References 26 publications

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

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

Active site acidic residues and structural analysis of modelled human aromatase: A potential drug target for breast cancer

X-ray structure of human aromatase reveals an androgen-specific active site

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