Emily E Scott

The Scripps Research Institute, La Jolla, California, United States

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Publications (28)155.56 Total impact

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    ABSTRACT: Crystallographic studies of different membrane cytochrome P450 enzymes have provided examples of distinct structural conformations, suggesting protein flexibility. It has been speculated that conformational selection is an integral component of substrate recognition and access, but direct evidence of such substate interconversion has thus far remained elusive. In the current study, solution NMR revealed multiple and exchanging backbone conformations for certain structural features of the human steroidogenic cytochrome P450 17A1 (CYP17A1). This bi-functional enzyme is responsible for pregnenolone C17 hydroxylation, followed by a 17,20-lyase reaction to produce dehydroepiandrosterone, the key intermediate in human synthesis of androgen and estrogen sex steroids. The distribution of CYP17A1 conformational states was influenced by temperature, binding of these two substrates, as well as binding of the soluble domain of cytochrome b5 (b5). Notably, titration of b5 to CYP17A1/pregnenolone induced a set of conformational states closely resembling those of CYP17A1/17α-hydroxypregnenolone without b5, providing structural evidence consistent with the reported ability of b5 to selectively enhance 17,20-lyase activity. Solution NMR thus revealed a set of conformations likely to modulate human steroidogenesis by CYP17A1, demonstrating this approach has the potential to make similar contributions to understanding the functions of other membrane P450 enzymes involved in drug metabolism and disease states.
    Journal of Biological Chemistry 03/2014; · 4.65 Impact Factor
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    ABSTRACT: This report summarizes a symposium sponsored by the American Society for Pharmacology and Experimental Therapeutics at Experimental Biology in April 2013. Presentations discussed the status of cytochrome P450 knowledge, emphasizing advances and challenges in relating structure with function and in applying this information to drug design. First, at least one structure of most major human drug-metabolizing P450 enzymes is known. However the flexibility of these active sites can limit the predictive value of one structure for other ligands. A second limitation is our coarse-grain understanding of P450 interactions with membranes, other P450 enzymes, NADPH-cytochrome P450 reductase, and cytochrome b 5. Recent work has examined differential P450 interactions with reductase in mixed P450 systems and P450:P450 complexes in reconstituted systems and cells, suggesting another level of functional control. In addition, protein NMR is a new approach to probe these protein/protein interactions, identifying interacting b5 and P450 surfaces, showing that b5 and reductase binding are mutually exclusive, and demonstrating ligand modulation of CYP17A1/b5 interactions. One desired outcome is the application of such information to control drug metabolism and/or design selective P450 inhibitors. A final presentation highlighted development of a CYP3A4 inhibitor that slows clearance of HIV drugs otherwise rapidly metabolized by CYP3A4. Although understanding P450 structure/function relationships is an ongoing challenge, translational advances will benefit from continued integration of existing and new biophysical approaches.
    Drug metabolism and disposition: the biological fate of chemicals 10/2013; · 3.74 Impact Factor
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    ABSTRACT: PURPOSE: 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), one of the most prevalent and procarcinogenic compounds in tobacco, is bioactivated by respiratory cytochrome P450 (CYP) 2A13, forming DNA adducts and initiating lung cancer. CYP2A13 inhibition offers a novel strategy for chemoprevention of tobacco-associated lung cancer. METHODS: Twenty-four analogs of a 4-benzylmorpholine scaffold identified by high throughput screening were evaluated for binding and inhibition of both functional human CYP2A enzymes, CYP2A13 and the 94%-identical hepatic CYP2A6, whose inhibition is undesirable. Thus, selectivity is a major challenge in compound design. RESULTS: A key feature resulting in CYP2A13-selective binding and inhibition was substitution at the benzyl ortho position, with three analogs being >25-fold selective for CYP2A13 over CYP2A6. CONCLUSIONS: Two such analogs were negative for genetic and hERG toxicities and metabolically stable in human lung microsomes, but displayed rapid metabolism in human liver and in mouse and rat lung and liver microsomes, likely due to CYP2B-mediated degradation. A specialized knockout mouse mimicking the human lung demonstrates compound persistence in lung and provides an appropriate test model. Compound delivered by inhalation may be effective in the lung but rapidly cleared otherwise, limiting systemic exposure.
    Pharmaceutical Research 06/2013; · 4.74 Impact Factor
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    ABSTRACT: The membrane heme protein cytochrome b5 (b5) can enhance, inhibit, or have no effect on cytochrome P450 (P450) catalysis, depending on the specific P450, substrate, and reaction conditions, but the structural basis remains unclear. Herein the interactions between the soluble domain of microsomal b5 and the catalytic domain of the bi-functional steroidogenic cytochrome P450 17A1 (CYP17A1) were investigated. CYP17A1 performs both steroid hydroxylation, which is unaffected by b5, and an androgen-forming lyase reaction which is facilitated 10-fold by b5. NMR chemical shift mapping of b5 titrations with CYP17A1 indicate that the interaction occurs in an intermediate exchange regime and identifies charged surface residues involved in the protein/protein interface. The role of these residues is confirmed by disruption of the complex upon mutagenesis of either the anionic b5 residues (E48 or E49) or the corresponding cationic CYP17A1 residues (R347, R358, or R449). Cytochrome b5 binding to CYP17A1 is also mutually exclusive with binding of NADPH-cytochrome P450 reductase (CPR). To probe the differential effects of b5 on the two CYP17A1-mediated reactions and thus communication between the superficial b5 binding site and the buried CYP17A1 active site, CYP17A1/b5 complex formation was characterized with either hydroxylase or lyase substrates bound to CYP17A1. Significantly, the CYP17A1/b5 interaction is stronger when the hydroxylase substrate pregnenolone is present in the CYP17A1 active site than when the lyase substrate 17α-hydroxypregnenolone is in the active site. These findings form the basis for a clearer understanding of this important interaction by directly measuring the reversible binding of the two proteins, providing evidence of communication between the CYP17A1 active site and the superficial proximal b5 binding site.
    Journal of Biological Chemistry 04/2013; · 4.65 Impact Factor
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    ABSTRACT: Cytochrome P450 (CYP) 1A1 is an extrahepatic monooxygenase involved in the metabolism of endogenous substrates and drugs, as well as the activation of certain toxins and environmental pollutants. CYP1A1 is particularly well known for its ability to biotransform polycyclic aromatic hydrocarbons (PAHs), such as benzo[a]pyrene in tobacco smoke, into carcinogens. CYP1A1 possesses functional similarities and differences with human CYP1A2 and CYP1B1 enzymes, but the structural basis for this has been unclear. We determined a 2.6 Å structure of human CYP1A1 with the inhibitor α-naphthoflavone (ANF). ANF binds within an enclosed active site, with the planar benzochromen-4-one core packed flat against the I helix that composes one wall of the active site, and the 2-phenyl substituent oriented toward the catalytic heme iron. Comparisons with previously determined structures of the related cytochrome P450 1A2 and 1B1 enzymes reveal distinct features among the active sites that may underlie the functional variability of these enzymes. Finally, docking studies probed the ability of CYP1A structures to assist in understanding their known in vitro interactions with several typical substrates and inhibitors.
    Journal of Biological Chemistry 03/2013; · 4.65 Impact Factor
  • Natasha M DeVore, Emily E Scott
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    ABSTRACT: Cytochromes P450 (CYP) from the 2A subfamily are known for their roles in the metabolism of nicotine, the addictive agent in tobacco, and activation of the tobacco procarcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Although both the hepatic CYP2A6 and respiratory CYP2A13 enzymes metabolize these compounds, CYP2A13 does so with much higher catalytic efficiency, but the structural basis for this has been unclear. X-ray structures of nicotine complexes with CYP2A13 (2.5 Å) and CYP2A6 (2.3 Å) yield a structural rationale for the preferential binding of nicotine to CYP2A13. Additional structures of CYP2A13 with NNK reveal either a single NNK molecule in the active site with orientations corresponding to metabolites known to form DNA adducts and initiate lung cancer (2.35 Å) or with two molecules of NNK bound (2.1 Å): one in the active site and one in a more distal staging site. Finally, in contrast to prior CYP2A structures with enclosed active sites, CYP2A13 conformations were solved that adopt both open and intermediate conformations resulting from an ∼2.5 Å movement of the F to G helices. This channel occurs in the same region where the second, distal NNK molecule is bound, suggesting that the channel may be used for ligand entry and/or exit from the active site. Altogether these structures provide multiple new snapshots of CYP2A13 conformations that assist in understanding the binding and activation of an important human carcinogen, as well as critical comparisons in the binding of nicotine, one of the most widely used and highly addictive drugs in human use.
    Journal of Biological Chemistry 06/2012; 287(32):26576-85. · 4.65 Impact Factor
  • Eva S Stephens, Agnes A Walsh, Emily E Scott
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    ABSTRACT: Cytochrome P450 (P450) enzymes are mixed-function oxidases that catalyze the metabolism of xenobiotics and endogenous biochemicals. Selective inhibitors are needed to accurately distinguish the contributions of individual P450 enzymes in the metabolism of drugs and the activation of procarcinogens in human tissues, but very frequently these enzymes have substantial overlapping selectivity. We evaluated a chemically diverse set of nine previously identified CYP2A6 inhibitors to determine which are able to discriminate between human CYP2A enzymes CYP2A6 and the 94%-identical CYP2A13 enzyme. Inhibitor binding to recombinant purified enzyme was evaluated, and affinities were determined. K(i) values were determined for inhibition of p-nitrophenol 2-hydroxylation, a reaction accomplished by CYP2A13 and CYP2A6 with more similar catalytic efficiencies (k(cat)/K(m) 0.19 and 0.12 μM⁻¹ · min⁻¹, respectively) than hydroxylation of the classic substrate coumarin (0.11 and 0.53 μM⁻¹ · min⁻¹, respectively). Of the nine compounds assayed, only tranylcypromine and (R)-(+)-menthofuran had a greater than 10-fold preference for CYP2A6 inhibition versus CYP2A13 inhibition. Most compounds evaluated [tryptamine, 4-dimethylaminobenzaldehyde, phenethyl isothiocyanate, β-nicotyrine, (S)-nicotine, and pilocarpine] demonstrated only moderate or no preference for inhibition of one CYP2A enzyme over the other. However, 8-methoxypsoralen has a 6-fold lower K(i) for CYP2A13 than for CYP2A6. This information is useful to inform reinterpretation of previous data with these inhibitors and to guide future studies seeking to determine which human CYP2A enzyme is responsible for the in vivo metabolism of compounds in human tissues expressing both enzymes.
    Drug metabolism and disposition: the biological fate of chemicals 06/2012; 40(9):1797-802. · 3.74 Impact Factor
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    Natasha M DeVore, Emily E Scott
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    ABSTRACT: Cytochrome P450 17A1 (also known as CYP17A1 and cytochrome P450c17) catalyses the biosynthesis of androgens in humans. As prostate cancer cells proliferate in response to androgen steroids, CYP17A1 inhibition is a new strategy to prevent androgen synthesis and treat lethal metastatic castration-resistant prostate cancer, but drug development has been hampered by lack of information regarding the structure of CYP17A1. Here we report X-ray crystal structures of CYP17A1, which were obtained in the presence of either abiraterone, a first-in-class steroidal inhibitor recently approved by the US Food and Drug Administration for late-stage prostate cancer, or TOK-001, an inhibitor that is currently undergoing clinical trials. Both of these inhibitors bind the haem iron, forming a 60° angle above the haem plane and packing against the central I helix with the 3β-OH interacting with aspargine 202 in the F helix. Notably, this binding mode differs substantially from those that are predicted by homology models and from steroids in other cytochrome P450 enzymes with known structures, and some features of this binding mode are more similar to steroid receptors. Whereas the overall structure of CYP17A1 provides a rationale for understanding many mutations that are found in patients with steroidogenic diseases, the active site reveals multiple steric and hydrogen bonding features that will facilitate a better understanding of the enzyme's dual hydroxylase and lyase catalytic capabilities and assist in rational drug design. Specifically, structure-based design is expected to aid development of inhibitors that bind only CYP17A1 and solely inhibit its androgen-generating lyase activity to improve treatment of prostate and other hormone-responsive cancers.
    Nature 02/2012; 482(7383):116-9. · 38.60 Impact Factor
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    ABSTRACT: Human xenobiotic-metabolizing cytochrome P450 (CYP) enzymes can each bind and monooxygenate a diverse set of substrates, including drugs, often producing a variety of metabolites. Additionally, a single ligand can interact with multiple CYP enzymes, but often the protein structural similarities and differences that mediate such overlapping selectivity are not well understood. Even though the CYP superfamily has a highly canonical global protein fold, there are large variations in the active site size, topology, and conformational flexibility. We have determined how a related set of three human CYP enzymes bind and interact with a common inhibitor, the muscarinic receptor agonist drug pilocarpine. Pilocarpine binds and inhibits the hepatic CYP2A6 and respiratory CYP2A13 enzymes much more efficiently than the hepatic CYP2E1 enzyme. To elucidate key residues involved in pilocarpine binding, crystal structures of CYP2A6 (2.4 Å), CYP2A13 (3.0 Å), CYP2E1 (2.35 Å), and the CYP2A6 mutant enzyme, CYP2A6 I208S/I300F/G301A/S369G (2.1 Å) have been determined with pilocarpine in the active site. In all four structures, pilocarpine coordinates to the heme iron, but comparisons reveal how individual residues lining the active sites of these three distinct human enzymes interact differently with the inhibitor pilocarpine.
    FEBS Journal 11/2011; 279(9):1621-31. · 4.25 Impact Factor
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    ABSTRACT: Histamine dehydrogenase (HADH) isolated from Nocardioides simplex catalyzes the oxidative deamination of histamine to imidazole acetaldehyde. HADH is highly specific for histamine, and we are interested in understanding the recognition mode of histamine in its active site. We describe the first crystal structure of a recombinant form of HADH (HADH) to 2.7-A resolution. HADH is a homodimer, where each 76-kDa subunit contains an iron-sulfur cluster ([4Fe-4S](2+)) and a 6-S-cysteinyl flavin mononucleotide (6-S-Cys-FMN) as redox cofactors. The overall structure of HADH is very similar to that of trimethylamine dehydrogenase (TMADH) from Methylotrophus methylophilus (bacterium W3A1). However, some distinct differences between the structure of HADH and TMADH have been found. Tyr(60), Trp(264), and Trp(355) provide the framework for the "aromatic bowl" that serves as a trimethylamine-binding site in TMADH is comprised of Gln(65), Trp(267), and Asp(358), respectively, in HADH. The surface Tyr(442) that is essential in transferring electrons to electron-transfer flavoprotein (ETF) in TMADH is not conserved in HADH. We use this structure to propose the binding mode for histamine in the active site of HADH through molecular modeling and to compare the interactions to those observed for other histamine-binding proteins whose structures are known.
    Journal of Biological Chemistry 08/2010; 285(33):25782-91. · 4.65 Impact Factor
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    ABSTRACT: Human microsomal cytochrome P450 (CYP) 2E1 is widely known for its ability to oxidize >70 different, mostly compact, low molecular weight drugs and other xenobiotic compounds. In addition CYP2E1 oxidizes much larger C9-C20 fatty acids that can serve as endogenous signaling molecules. Previously structures of CYP2E1 with small molecules revealed a small, compact CYP2E1 active site, which would be insufficient to accommodate medium and long chain fatty acids without conformational changes in the protein. In the current work we have determined how CYP2E1 can accommodate a series of fatty acid analogs by cocrystallizing CYP2E1 with omega-imidazolyl-octanoic fatty acid, omega-imidazolyl-decanoic fatty acid, and omega-imidazolyl-dodecanoic fatty acid. In each structure direct coordination of the imidazole nitrogen to the heme iron mimics the position required for native fatty acid substrates to yield the omega-1 hydroxylated metabolites that predominate experimentally. In each case rotation of a single Phe(298) side chain merges the active site with an adjacent void, significantly altering the active site size and topology to accommodate fatty acids. The binding of these fatty acid ligands is directly opposite the channel to the protein surface and the binding observed for fatty acids in the bacterial cytochrome P450 BM3 (CYP102A1) from Bacillus megaterium. Instead of the BM3-like binding mode in the CYP2E1 channel, these structures reveal interactions between the fatty acid carboxylates and several residues in the F, G, and B' helices at successive distances from the active site.
    Journal of Biological Chemistry 05/2010; 285(29):22282-90. · 4.65 Impact Factor
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    ABSTRACT: Prolyl 4-hydroxylases (P4H) catalyze the post-translational hydroxylation of proline residues and play a role in collagen production, hypoxia response, and cell wall development. P4Hs belong to the group of Fe(II)/alphaKG oxygenases and require Fe(II), alpha-ketoglutarate (alphaKG), and O(2) for activity. We report the 1.40 A structure of a P4H from Bacillus anthracis, the causative agent of anthrax, whose immunodominant exosporium protein BclA contains collagen-like repeat sequences. The structure reveals the double-stranded beta-helix core fold characteristic of Fe(II)/alphaKG oxygenases. This fold positions Fe-binding and alphaKG-binding residues in what is expected to be catalytically competent orientations and is consistent with proline peptide substrate binding at the active site mouth. Comparisons of the anthrax P4H structure with Cr P4H-1 structures reveal similarities in a peptide surface groove. However, sequence and structural comparisons suggest differences in conformation of adjacent loops may change the interaction with peptide substrates. These differences may be the basis of a substantial disparity between the K(M) values for the Cr P4H-1 compared to the anthrax and human P4H enzymes. Additionally, while previous structures of P4H enzymes are monomers, B. anthracis P4H forms an alpha(2) homodimer and suggests residues important for interactions between the alpha(2) subunits of alpha(2)beta(2) human collagen P4H. Thus, the anthrax P4H structure provides insight into the structure and function of the alpha-subunit of human P4H, which may aid in the development of selective inhibitors of the human P4H enzyme involved in fibrotic disease.
    Biochemistry 11/2009; 49(1):124-33. · 3.38 Impact Factor
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    ABSTRACT: Although the human lung cytochrome P450 2A13 (CYP2A13) and its liver counterpart cytochrome P450 2A6 (CYP2A6) are 94% identical in amino acid sequence, they metabolize a number of substrates with substantially different efficiencies. To determine differences in binding for a diverse set of cytochrome P450 2A ligands, we have measured the spectral binding affinities (K(D)) for nicotine, phenethyl isothiocyanate (PEITC), coumarin, 2'-methoxyacetophenone (MAP), and 8-methoxypsoralen. The differences in the K(D) values for CYP2A6 versus CYP2A13 ranged from 74-fold for 2'-methoxyacetophenone to 1.1-fold for coumarin, with CYP2A13 demonstrating the higher affinity. To identify active site amino acids responsible for the differences in binding of MAP, PEITC, and coumarin, 10 CYP2A13 mutant proteins were generated in which individual amino acids from the CYP2A6 active site were substituted into CYP2A13 at the corresponding position. Titrations revealed that substitutions at positions 208, 300, and 301 individually had the largest effects on ligand binding. The collective relevance of these amino acids to differential ligand selectivity was verified by evaluating binding to CYP2A6 mutant enzymes that incorporate several of the CYP2A13 amino acids at these positions. Inclusion of four CYP2A13 amino acids resulted in a CYP2A6 mutant protein (I208S/I300F/G301A/S369G) with binding affinities for MAP and PEITC much more similar to those observed for CYP2A13 than to those for CYP2A6 without altering coumarin binding. The structure-based quantitative structure-activity relationship analysis using COMBINE successfully modeled the observed mutant-ligand trends and emphasized steric roles for active site residues including four substituted amino acids and an adjacent conserved Leu(370).
    Drug metabolism and disposition: the biological fate of chemicals 03/2009; 37(6):1319-27. · 3.74 Impact Factor
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    ABSTRACT: Human microsomal cytochrome P-450 2E1 (CYP2E1) monooxygenates > 70 low molecular weight xenobiotic compounds, as well as much larger endogenous fatty acid signaling molecules such as arachidonic acid. In the process, CYP2E1 can generate toxic or carcinogenic compounds, as occurs with acetaminophen overdose, nitrosamines in cigarette smoke, and reactive oxygen species from uncoupled catalysis. Thus, the diverse roles that CYP2E1 has in normal physiology, toxicity, and drug metabolism are related to its ability to metabolize diverse classes of ligands, but the structural basis for this was previously unknown. Structures of human CYP2E1 have been solved to 2.2 angstroms for an indazole complex and 2.6 angstroms for a 4-methylpyrazole complex. Both inhibitors bind to the heme iron and hydrogen bond to Thr303 within the active site. Complementing its small molecular weight substrates, the hydrophobic CYP2E1 active site is the smallest yet observed for a human cytochrome P-450. The CYP2E1 active site also has two adjacent voids: one enclosed above the I helix and the other forming a channel to the protein surface. Minor repositioning of the Phe478 aromatic ring that separates the active site and access channel would allow the carboxylate of fatty acid substrates to interact with conserved 216QXXNN220 residues in the access channel while positioning the hydrocarbon terminus in the active site, consistent with experimentally observed omega-1 hydroxylation of saturated fatty acids. Thus, these structures provide insights into the ability of CYP2E1 to effectively bind and metabolize both small molecule substrates and fatty acids.
    Journal of Biological Chemistry 10/2008; 283(48):33698-707. · 4.65 Impact Factor
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    ABSTRACT: Histamine dehydrogenase (HADH) from Nocardioides simplex catalyzes the oxidative deamination of histamine to produce imidazole acetaldehyde and an ammonium ion. HADH is functionally related to trimethylamine dehydrogenase (TMADH), but HADH has strict substrate specificity towards histamine. HADH is a homodimer, with each 76 kDa subunit containing two redox cofactors: a [4Fe-4S] cluster and an unusual covalently bound flavin mononucleotide, 6-S-cysteinyl-FMN. In order to understand the substrate specificity of HADH, it was sought to determine its structure by X-ray crystallography. This enzyme has been expressed recombinantly in Escherichia coli and successfully crystallized in two forms. Diffraction data were collected to 2.7 A resolution at the SSRL synchrotron with 99.7% completeness. The crystals belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 101.14, b = 107.03, c = 153.35 A.
    Acta Crystallographica Section F Structural Biology and Crystallization Communications 10/2008; 64(Pt 9):785-7. · 0.55 Impact Factor
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    ABSTRACT: Cytochrome P450s (P450s) metabolize a large number of diverse substrates with specific regio- and stereospecificity. A number of compounds, including nicotine, cotinine, and aflatoxin B(1), are metabolites of the 94% identical CYP2A13 and CYP2A6 enzymes but at different rates. Phenacetin and 4-aminobiphenyl were identified as substrates of human cytochromes P450 1A2 and 2A13 but not of CYP2A6. The purpose of this study was to identify active site amino acids that are responsible for CYP2A substrate specificity using phenacetin as a structural probe. Ten amino acid residues that differ in the CYP2A13 and CYP2A6 active sites were exchanged between the two enzymes. Phenacetin binding revealed that the six substitution, CYP2A13 S208I, A213S, F300I, A301G, M365V, and G369S decreased phenacetin affinity. Although incorporation of individual CYP2A13 residues into CYP2A6 had little effect on this enzyme's very low levels of phenacetin metabolism, the combination of double, triple, and quadruple substitutions at positions 208, 300, 301, and 369 increasingly endowed CYP2A6 with the ability to metabolize phenacetin. Enzyme kinetics revealed that the CYP2A6 I208S/I300F/G301A/S369G mutant protein O-deethylated phenacetin with a K(m) of 10.3 muM and a k(cat) of 2.9 min(-1), which compare very favorably with those of CYP2A13 (K(m) of 10.7 muM and k(cat) of 3.8 min(-1)). A 2.15 A crystal structure of the mutant CYP2A6 I208S/I300F/G301A/S369G protein with phenacetin in the active site provided a structural rationale for the differences in phenacetin metabolism between CYP2A6 and CYP2A13.
    Drug metabolism and disposition: the biological fate of chemicals 10/2008; 36(12):2582-90. · 3.74 Impact Factor
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    Megen A Miller, Emily E Scott, Julian Limburg
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    ABSTRACT: Collagen prolyl-4-hydroxylase (C-P4H) catalyzes the hydroxylation of specific proline residues in procollagen, which is an essential step in collagen biosynthesis. A new form of P4H from Bacillus anthracis (anthrax-P4H) that shares many characteristics with the type I C-P4H from human has recently been characterized. The structure of anthrax-P4H could provide important insight into the chemistry of C-P4Hs and into the function of this unique homodimeric P4H. X-ray diffraction data of selenomethionine-labeled anthrax-P4H recombinantly expressed in Escherichia coli have been collected to 1.4 A resolution.
    Acta Crystallographica Section F Structural Biology and Crystallization Communications 10/2008; 64(Pt 9):788-91. · 0.55 Impact Factor
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    ABSTRACT: Cytochrome P450 2A13 (CYP2A13) is a lung specific enzyme known to activate the potent tobacco procarcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) into two carcinogenic metabolites. CYP2A13 has been crystallized and X-ray diffraction experiments illuminated the structure of this enzyme, but with an unknown ligand present in the enzyme active site. This unknown ligand was suspected to be indole but a selective method had to be developed to differentiate among indole and its metabolites in the protein sample. We successfully modified a microbiological colorimetric assay to spectrophotometrically differentiate between indole and a number of possible indole metabolites in nanomolar concentrations by derivatization with p-dimethylaminocinnamaldehyde (DMACA). Further differentiation of indoles was made by mass spectrometry (HPLC-UV/vis-MS/MS) utilizing the chromophore generated in the DMACA conjugation as a UV signature for HPLC detection. The ligand in the crystallized protein was identified as unsubstituted indole, which facilitated refinement of two alternate conformations in the CYP2A13 crystal structure active site.
    Archives of Biochemistry and Biophysics 08/2008; 475(1):14-7. · 3.37 Impact Factor
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    ABSTRACT: CYP2A13 is an efficient catalyst of metabolic activation of the human carcinogens 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN). This study investigated the functional consequences of CYP2A13 polymorphisms that result in single amino acid substitutions. Five CYP2A13 variants, namely CYP2A13*2 (R257C), CYP2A13*5 (F453Y), CYP2A13*6 (R494C), CYP2A13*8 (D158E), and CYP2A13*9 (V323L), were expressed and evaluated for coumarin binding affinity, coumarin 7-hydroxylation, and -hydroxylation of (S)-NNN and NNK. In addition, the 133_134 Thr deletion variant, coded for by CYP2A13*3, was expressed but was not stable to the protein purification procedure. A 30-42% decrease in coumarin 7-hydroxylation catalytic efficiency was determined for R257C and D158E. No effect on coumarin binding or (S)-NNN metabolism was observed. Three variants, R257C, D158E, and V323L, had two- to threefold decreased catalytic efficiency for NNK -hydroxylation. CYP2A13 polymorphisms resulted in modest changes in coumarin 7-hydroxylation and NNK -hydroxylation activities in vitro. Although these changes are not likely to impact in vivo metabolism, these data should aid in the interpretation and design of future epidemiology studies.
    Xenobiotica 01/2008; 37(12):1439-49. · 1.98 Impact Factor
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    ABSTRACT: The human lung cytochrome P450 2A13 (CYP2A13) activates the nicotine-derived procarcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) into DNA-altering compounds that cause lung cancer. Another cytochrome P450, CYP2A6, is also present in human lung, but at much lower levels. Although these two enzymes are 93.5% identical, CYP2A13 metabolizes NNK with much lower K(m) values than does CYP2A6. To investigate the structural differences between these two enzymes the structure of CYP2A13 was determined to 2.35A by x-ray crystallography and compared with structures of CYP2A6. As expected, the overall CYP2A13 and CYP2A6 structures are very similar with an average root mean square deviation of 0.5A for the Calpha atoms. Like CYP2A6, the CYP2A13 active site cavity is small and highly hydrophobic with a cluster of Phe residues composing the active site roof. Active site residue Asn(297) is positioned to hydrogen bond with an adventitious ligand, identified as indole. Amino acid differences between CYP2A6 and CYP2A13 at positions 117, 300, 301, and 208 relate to different orientations of the ligand plane in the two protein structures and may underlie the significant variations observed in binding and catalysis of many CYP2A ligands. In addition, docking studies suggest that residues 365 and 366 may also contribute to differences in NNK metabolism.
    Journal of Biological Chemistry 07/2007; 282(23):17306-13. · 4.65 Impact Factor

Publication Stats

684 Citations
155.56 Total Impact Points


  • 2013
    • The Scripps Research Institute
      La Jolla, California, United States
  • 2005–2013
    • University of Kansas
      • Department of Medicinal Chemistry
      Lawrence, Kansas, United States
  • 2002–2005
    • University of Texas Medical Branch at Galveston
      • Department of Pharmacology and Toxicology
      Galveston, TX, United States