F Peter Guengerich

Vanderbilt University, Нашвилл, Michigan, United States

Are you F Peter Guengerich?

Claim your profile

Publications (917)3918.92 Total impact

  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Aspergillus fumigatus is the opportunistic fungal pathogen that predominantly affects the immunocompromised population and causes 600,000 deaths per year. The cytochrome P450 (CYP) 51 inhibitor voriconazole is currently the drug of choice, yet the treatment efficiency remains low, calling for rational development of more efficient agents. A. fumigatus has two CYP51 genes, CYP51A and CYP51B, which share 59% amino acid sequence identity. CYP51B is expressed constitutively, while gene CYP51A is reported to be inducible. We expressed, purified, and characterized A. fumigatus CYP51B, including determination of its substrate preferences, catalytic parameters, inhibition, and X-ray structure in complexes with voriconazole and the experimental inhibitor (R)-N-(1-(2,4-dichlorophenyl)-2-(1H-imidazol-1-yl)ethyl)-4-(5-phenyl-1,3,4-oxadiazol-2-yl)benzamide (VNI). The enzyme demethylated its natural substrate eburicol and the plant CYP51 substrate obtusifoliol at steady-state rates of 17 and 16 min-1, respectively, but did not metabolize lanosterol, and the topical antifungal drug miconazole was the strongest inhibitor that we identified. The X-ray crystal structures displayed high overall similarity of A. fumigatus CYP51B to CYP51 orthologs from other biological kingdoms but revealed phylum-specific differences relevant to enzyme catalysis and inhibition. The complex with voriconazole provides an explanation for the potency of this relatively small molecule, while the complex with VNI outlines a direction for further enhancement of the efficiency of this new inhibitory scaffold to treat humans afflicted with filamentous fungal infections. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
    Journal of Biological Chemistry 08/2015; DOI:10.1074/jbc.M115.677310 · 4.57 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: 1. The polycyclic hydrocarbons (PAHs), pyrene, 1-hydroxypyrene, 1-nitropyrene and 1-acetylpyrene, were found to induce Type I binding spectra with human cytochrome P450 (P450) 2A13 and were converted to various mono- and di-oxygenated products by this enzyme. 2. Pyrene was first oxidized by P450 2A13 to 1-hydroxypyrene which was further oxidized to di-oxygenated products, i.e. 1,8- and 1,6-dihydroxypyrene. Of five other human P450s examined, P450 1B1 catalyzed pyrene oxidation to 1-hydroxypyrene at a similar rate to P450 2A13 but was less efficient in forming dihydroxypyrenes. P450 2A6, a related human P450 enzyme, which did not show any spectral changes with these four PAHs, showed lower activities in oxidation of these compounds than P450 2A13. 3. 1-Nitropyrene and 1-acetylpyrene were also found to be efficiently oxidized by P450 2A13 to several oxygenated products, based on mass spectrometry analysis. 4. Molecular docking analysis supported preferred orientations of pyrene and its derivatives in the active site of P450 2A13, with lower interaction energies (U values) than observed for P450 2A6 and that several amino acid residues (including Ala-301, Asn-297 and Ala-117) play important roles in directing the orientation of these PAHs in the P450 2A13 active site. In addition, Phe-231 and Gly-329 were found to interact with pyrene to orient this compound in the active site of P450 1B1. 5. These results suggest that P450 2A13 is one of the important enzymes that oxidizes these PAH compounds and may determine how these chemicals are detoxicated and bioactivated in humans.
    Xenobiotica 08/2015; · 2.20 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Cytochrome P450 21A2 is a key player in steroid 21-hydroxylation and converts progesterone to 11-deoxycorticosterone and 17α-hydroxy-progesterone to 11-deoxycortisol. More than 100 mutations in P450 21A2 have been established in patients thus far; these account for the vast majority of occurrences of congenital adrenal hyperplasia (CAH), which is among the most common heritable metabolic diseases in humans. CAH phenotypes range from the most severe, salt-wasting (SW), to the simple virilizing (SV) and nonclassical (NC) CAH forms. We recently determined the crystal structure of human P450 21A2 in complex with progesterone. To gain more insight into the structural and stability changes underlying the phenotypes of individual mutations, we analyzed 24 SW, SV, and NC mutants in the context of the crystal structure of the human enzyme. Our analysis reveals clear differences in the localization of SW, SV and NC mutations, with many of the first type mapping to the active site and near the heme and/or substrate and mostly resulting in complete loss of enzyme activity. Conversely, NC mutations are often found near the periphery and close to the surface of the protein, and mutant enzymes retain partial activity. The main conclusion from the mutation-structure-activity study is that the severity of the CAH clinical manifestations can be directly correlated with the degree of mutation-induced damage in terms of protein fold stability and active site changes in the structural model. Thus, the NC phenotype is typically associated with mutations that have a compensatory effect, i.e. H-bonding replacing hydrophobic interactions and vice versa.
    Molecular Endocrinology 07/2015; 29(9):ME20151127. DOI:10.1210/ME.2015-1127 · 4.02 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Morphine, first characterized in opium from the poppy Papaver somniferum, is one of the strongest known analgesics. Endogenous morphine has been identified in several mammalian cells and tissues. The synthetic pathway of morphine in the opium poppy has been elucidated, and the presence of common intermediates in plants and mammals suggests that biosynthesis occurs through similar pathways (beginning with the amino acid L-tyrosine), and the pathway has been completely delineated in plants. Some of the enzymes in the mammalian pathway have been identified and characterized. Two of the latter steps in the morphine biosynthesis pathway are demethylation of thebaine at the O3- and the O6-positions, the latter of which has been difficult to demonstrate. The plant enzymes responsible for both the O3- and the O6-demethylation are members of the FeII/α-ketoglutarate-dependent dioxygenase family. Previous studies showed that human cytochrome P450 (P450) 2D6 can catalyze thebaine O3-demethylation. We report that demethylation of thebaine at the O6-position is selectively catalyzed by human P450s 3A4 and 3A5, with the latter being more efficient, and rat P450 3A2. Our results do not support O6-demethylation of thebaine by an FeII/α-ketoglutarate-dependent dioxygenase. In rat brain microsomes, O6-demethylation was inhibited by ketoconazole, but not sulfaphenazole, suggesting that P450 3A enzymes are responsible for this activity in the brain. An alternate pathway to morphine, oripavine O6-demethylation, was not detected. The major enzymatic steps in mammalian morphine synthesis have now been identified. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
    Journal of Biological Chemistry 07/2015; 290(33). DOI:10.1074/jbc.M115.665331 · 4.57 Impact Factor
  • F Peter Guengerich
    [Show abstract] [Hide abstract]
    ABSTRACT: Four Minireviews deal with aspects of the α-ketoglutarate/iron dependent dioxygenases in this eighth Thematic Series on Metals in Biology. The Minireviews cover a general introduction and synopsis of the current understanding of mechanisms of catalysis, the roles of these dioxygenases in post-translational protein modification and de-modification, the roles of the ten-eleven translocation (Tet) dioxygenases in the modification of methylated bases (5mC, T) in DNA relevant to epigenetic mechanisms, and the roles of the AlkB-related dioxygenases in the repair of damaged DNA and RNA. The use of α-ketoglutarate (alternatively termed 2-oxoglutarate) as a co-substrate in so many oxidation reactions throughout much of nature is notable and has surprisingly emerged from biochemical and genomic analysis. About 60 of these enzymes are now recognized in humans, and a number have been identified as having critical functions. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
    Journal of Biological Chemistry 07/2015; 290(34). DOI:10.1074/jbc.R115.675652 · 4.57 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: N6-(2-Hydroxy-3-buten-1-yl)-2'-deoxyadenosine (N6-HB-dA I) and N6,N6-(2,3-dihydroxybutan-1,4-diyl)-2'-deoxyadenosine (N6,N6-DHB-dA) are exocyclic DNA adducts formed upon alkylation of the N6 position of adenine in DNA by epoxide metabolites of 1,3-butadiene (BD), a common industrial and environmental chemical classified as a human and animal carcinogen. Since the N6-H atom of adenine is required for Watson-Crick hydrogen bonding with thymine, N6-alkylation can prevent adenine from normal pairing with thymine, potentially compromising the accuracy of DNA replication. To evaluate the ability of BD-derived N6-alkyladenine lesions to induce mutations, synthetic oligodeoxynucleotides containing site-specific (S)-N6-HB-dA I and (R,R)-N6,N6-DHB-dA adducts were subjected to in vitro translesion synthesis in the presence of human DNA polymerases β, η, ι, and κ. While (S)-N6-HB-dA I was readily bypassed by all four enzymes, only polymerases η and κ were able to carry out DNA synthesis past (R,R)-N6,N6-DHB-dA. Steady-state kinetic analyses indicated that all four DNA polymerases preferentially incorporated the correct base (T) opposite (S)-N6-HB-dA I. In contrast, hPol β was completely blocked by (R,R)-N6,N6-DHB-dA, while hPol η and κ inserted A, G, C, or T opposite the adduct with similar frequency. HPLC-ESI-MS/MS analysis of primer extension products confirmed that while translesion synthesis past (S)-N6-HB-dA I was mostly error-free, replication of DNA containing (R,R)-N6,N6-DHB-dA induced significant numbers of A, C, and G insertions and small deletions. These results indicate that singly substituted (S)-N6-HB-dA I lesions are not miscoding, but exocyclic (R,R)-N6,N6-DHB-dA adducts are strongly mispairing, probably due to their inability to form stable Watson-Crick pairs with dT.
    Chemical Research in Toxicology 06/2015; 28(7). DOI:10.1021/acs.chemrestox.5b00166 · 3.53 Impact Factor
  • F Peter Guengerich
    [Show abstract] [Hide abstract]
    ABSTRACT: This seventh Metals in Biology Thematic Series deals with the metal-based interactions of mammalian hosts with pathogens. Both pathogens and host have complex regulatory systems for metal homeostasis. Understanding these provides strategies for fighting pathogens, either by excluding essential metals from the microbes, by delivery of excess metals to cause toxicity, or by complexing metals in microorganisms. Intervention is possible by delivery of complexing reagents or by targeting the microbial regulatory apparatus. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
    Journal of Biological Chemistry 06/2015; 290(31). DOI:10.1074/jbc.R115.670265 · 4.57 Impact Factor
  • Source
    Yan Su · Amritraj Patra · Joel M Harp · Martin Egli · F Peter Guengerich
    [Show abstract] [Hide abstract]
    ABSTRACT: Like the other Y-Family DNA polymerases, human DNA polymerase η (hpol η) has relatively low fidelity and is able to tolerate damage during DNA synthesis, including 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxoG), one of the most abundant DNA lesions in the genome. Crystal structures show that Arg-61 and Gln-38 are located near the active site and may play important roles in the fidelity and efficiency of hpol η. Site-directed mutagenesis was used to replace these side chains either alone or together, and the wild type or mutant proteins were purified and tested by replicating DNA past deoxyguanosine (G) or 8-oxoG. The catalytic activity of hpol η was dramatically disrupted by the R61M and Q38A/R61A mutations, as opposed to the R61A and Q38A single mutants. Crystal structures of hpol η mutant ternary complexes reveal that polarized water molecules can mimic and partially compensate for the missing side chains of Arg-61 and Gln-38 in the Q38A/R61A mutant. The combined data indicate that the positioning and positive charge of Arg-61 synergistically contribute to the nucleotidyl transfer reaction, with additional influence exerted by Gln-38. In addition, gel filtration chromatography separated multimeric and monomeric forms of wild type and mutant hpol η, indicating the possibility that hpol η forms multimers in vivo. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
    Journal of Biological Chemistry 05/2015; 290(26). DOI:10.1074/jbc.M115.653691 · 4.57 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Cytochrome P450 (P450) 21A2 is the major steroid 21-hydroxylase, and deficiency of this enzyme is involved in ~ 95% of cases of human congenital adrenal hyperplasia, a disorder of adrenal steroidogenesis. A structure of the bovine enzyme we previously published (Zhao, B. et al. (2012) J. Biol. Chem. 287, 10613-10622), containing two molecules of the substrate 17alpha-hydroxyprogesterone, has been used as a template for understanding genetic deficiencies. We have now obtained a crystal structure of human P450 21A2 in complex with progesterone, a substrate in adrenal 21-hydroxylation. Substrate binding and release were fast for human P450 21A2 with both substrates, and pre-steady-state kinetics showed a partial burst but only with progesterone as substrate and not 17alpha-hydroxyprogesterone. High intermolecular non-competitive kinetic deuterium isotope effects on both kcat and kcat/Km (5-11) were observed with both substrates, indicative of rate-limiting C-H bond cleavage and suggesting that the juxtaposition of the C21 carbon in the active site is critical for efficient oxidation. The estimated rate of binding of the substrate progesterone (kon 2.4 x 107 M-1 s-1) is only ~ 2-fold greater than the catalytic efficiency (kcat/Km 1.3 x 107 M-1 s-1) with this substrate, suggesting that the rate of substrate binding may also be partially rate-limiting. The structure of the human P450 21A2-substrate complex provides direct insight into mechanistic effects of genetic variants. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
    Journal of Biological Chemistry 04/2015; 290(21). DOI:10.1074/jbc.M115.646307 · 4.57 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Etheno DNA adducts are a prevalent type of DNA damage caused by vinyl chloride (VC) exposure and oxidative stress. Etheno adducts are mutagenic and may contribute to the initiation of several pathologies; thus, elucidating the pathways by which they induce cellular transformation is critical. Although N(2),3-ethenoguanine (N(2),3-εG) is the most abundant etheno adduct, its biological consequences have not been well characterized in cells due to its labile glycosidic bond. Here, a stabilized 2'-fluoro-2'-deoxyribose analog of N(2),3-εG was used to quantify directly its genotoxicity and mutagenicity. A multiplex method involving next-generation sequencing enabled a large-scale in vivo analysis, in which both N(2),3-εG and its isomer 1,N(2)-ethenoguanine (1,N(2)-εG) were evaluated in various repair and replication backgrounds. We found that N(2),3-εG potently induces G to A transitions, the same mutation previously observed in VC-associated tumors. By contrast, 1,N(2)-εG induces various substitutions and frameshifts. We also found that N(2),3-εG is the only etheno lesion that cannot be repaired by AlkB, which partially explains its persistence. Both εG lesions are strong replication blocks and DinB, a translesion polymerase, facilitates the mutagenic bypass of both lesions. Collectively, our results indicate that N(2),3-εG is a biologically important lesion and may have a functional role in VC-induced or inflammation-driven carcinogenesis. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.
    Nucleic Acids Research 04/2015; 43(11). DOI:10.1093/nar/gkv243 · 9.11 Impact Factor
  • Mirza Bojić · Carl A Sedgeman · Leslie D Nagy · F. Peter Guengerich
    [Show abstract] [Hide abstract]
    ABSTRACT: Aspirin (acetylsalicylic acid) is a well-known and widely-used analgesic. It is rapidly deacetylated to salicylic acid, which forms two hippuric acids-salicyluric acid and gentisuric acid-and two glucuronides. The oxidation of aspirin and salicylic acid has been reported with human liver microsomes, but data on individual cytochromes P450 involved in oxidation is lacking. In this study we monitored oxidation of these compounds by human liver microsomes and cytochrome P450 (P450) using UPLC with fluorescence detection. Microsomal oxidation of salicylic acid was much faster than aspirin. The two oxidation products were 2,5-dihydroxybenzoic acid (gentisic acid, documented by its UV and mass spectrum) and 2,3-dihydroxybenzoic acid. Formation of neither product was inhibited by desferrioxamine, suggesting a lack of contribution of oxygen radicals under these conditions. Although more liphophilic, aspirin was oxidized less efficiently, primarily to the 2,5-dihydroxy product. Recombinant human P450s 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4 all catalyzed the 5-hydroxylation of salicylic acid. Inhibitor studies with human liver microsomes indicated that all six of the previously mentioned P450s could contribute to both the 5- and 3-hydroxylation of salicylic acid and that P450s 2A6 and 2B6 have contributions to 5-hydroxylation. Inhibitor studies indicated that the major human P450 involved in both 3- and 5-hydroxylation of salicylic acid is P450 2E1. Copyright © 2015 Elsevier B.V. All rights reserved.
    European journal of pharmaceutical sciences: official journal of the European Federation for Pharmaceutical Sciences 03/2015; 73. DOI:10.1016/j.ejps.2015.03.015 · 3.35 Impact Factor
  • Goutam Chowdhury · F Peter Guengerich
    [Show abstract] [Hide abstract]
    ABSTRACT: This unit contains a complete procedure for the detection and structural characterization of DNA protein crosslinks (DPCs). The procedure also describes an approach for the quantitation of the various structurally distinct DPCs. Although various methods have been described in the literature for labile DPCs, characterization of nonlabile adducts remain a challenge. Here we present a novel approach for characterization of both labile and non-labile adducts by the use of a combination of chemical, enzymatic, and mass spectrometric approaches. A Raney Ni-catalyzed reductive desulfurization method was used for removal of the bulky peptide adducts, enzymatic digestion was used to digest the protein to smaller peptides and DNA to nucleosides, and finally LC-ESI-tandem mass spectrometry (MS) was utilized for detection and characterization of nucleoside adducts. © 2015 by John Wiley & Sons, Inc. Copyright © 2015 John Wiley & Sons, Inc.
    Current protocols in nucleic acid chemistry / edited by Serge L. Beaucage ... [et al.] 03/2015; 60:10.15.1-10.15.14. DOI:10.1002/0471142700.nc1015s60
  • [Show abstract] [Hide abstract]
    ABSTRACT: AlkB proteins are evolutionary conserved Fe(II)/2-oxoglutarate-dependent dioxygenases, which remove alkyl and highly promutagenic etheno(ɛ)-DNA adducts, but their substrate specificity has not been fully determined. We developed a novel assay for the repair of ɛ-adducts by AlkB enzymes using oligodeoxynucleotides with a single lesion and specific DNA glycosylases and AP-endonuclease for identification of the repair products. We compared the repair of three ɛ-adducts, 1,N(6)-ethenoadenine (ɛA), 3,N(4)-ethenocytosine (ɛC) and 1,N(2)-ethenoguanine (1,N(2)-ɛG) by nine bacterial and two human AlkBs, representing four different structural groups defined on the basis of conserved amino acids in the nucleotide recognition lid, engaged in the enzyme binding to the substrate. Two bacterial AlkB proteins, MT-2B (from Mycobacterium tuberculosis) and SC-2B (Streptomyces coelicolor) did not repair these lesions in either double-stranded (ds) or single-stranded (ss) DNA. Three proteins, RE-2A (Rhizobium etli), SA-2B (Streptomyces avermitilis), and XC-2B (Xanthomonas campestris) efficiently removed all three lesions from the DNA substrates. Interestingly, XC-2B and RE-2A are the first AlkB proteins shown to be specialized for ɛ-adducts, since they do not repair methylated bases. Three other proteins, EcAlkB (Escherichia coli), SA-1A, and XC-1B removed ɛA and ɛC from ds and ssDNA but were inactive toward 1,N(2)-ɛG. SC-1A repaired only ɛA with the preference for dsDNA. The human enzyme ALKBH2 repaired all three ɛ-adducts in dsDNA, while only ɛA and ɛC in ssDNA and repair was less efficient in ssDNA. ALKBH3 repaired only ɛC in ssDNA. Altogether, we have shown for the first time that some AlkB proteins, namely ALKBH2, RE-2A, SA-2B and XC-2B can repair 1,N(2)-ɛG and that ALKBH3 removes only ɛC from ssDNA. Our results also suggest that the nucleotide recognition lid is not the sole determinant of the substrate specificity of AlkB proteins. Copyright © 2015. Published by Elsevier B.V.
    DNA repair 03/2015; 30. DOI:10.1016/j.dnarep.2015.02.021 · 3.11 Impact Factor
  • Source
    Amritraj Patra · Qianqian Zhang · Li Lei · Yan Su · Martin Egli · F Peter Guengerich
    [Show abstract] [Hide abstract]
    ABSTRACT: The most common lesion in DNA is an abasic site resulting from glycolytic cleavage of a base. In a number of cellular studies, abasic sites preferentially code for dATP insertion (the A Rule). In some cases frameshifts are also common. X-ray structures with abasic sites in oligonucleotides have been reported for several microbial and human DNA polymerases (pols), e.g. Dpo4, RB69, KlenTaq, yeast pol ι, human (h) pol ι, and human pol β. We previously reported that hpol η is a major pol involved in abasic site bypass (Choi, J-Y. et al. (2010) J. Mol. Biol. 404, 34-44). hPol η inserted all four dNTPs in steady-state and pre-steady-state assays, preferentially inserting A and G. In LC-MS analysis of primer-template pairs, A and G were inserted but little C or T was. Frameshifts were observed when an appropriate pyrimidine was positioned 5 to the abasic site in the template. In X-ray structures of hpol η with a non-hydrolyzable analog of dATP or dGTP opposite an abasic site, H-bonding was observed between the phosphate 5 to the abasic site and water H-bonded to N1 and N6 of A and N1 and O6 of G nucleoside triphosphate analogs, offering an explanation for what appears to be a purine rule. A structure was also obtained for an A inserted and bonded in the primer opposite the abasic site, but it did not pair with a 5 T in the template. We conclude that hpol η, a major copying enzyme with abasic sites, follows a purine rule, which can also lead to frameshifts. The phenomenon can be explained with H-bonds. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
    Journal of Biological Chemistry 02/2015; 290(13). DOI:10.1074/jbc.M115.637561 · 4.57 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Acenaphthene and acenaphthylene, two known environmental polycyclic aromatic hydrocarbon (PAH) pollutants, were incubated at 50 μM concentrations in a standard reaction mixture with human P450s 2A6, 2A13, 1B1, 1A2, 2C9, and 3A4, and the oxidation products were determined using HPLC and LC-MS. HPLC analysis showed that P450 2A6 converted acenaphthene and acenaphthylene to several mono- and dioxygenated products. LC-MS analysis of acenaphthene oxidation by P450s indicated the formation of 1-acenaphthenol as a major product, with turnover rates of 6.7, 4.5, and 3.6 nmol product formed/min/nmol P450 for P450 2A6, 2A13, and 1B1, respectively. Acenaphthylene oxidation by P450 2A6 showed the formation of 1,2-epoxyacenaphthene as a major product (4.4 nmol epoxide formed/min/nmol P450) and also several mono- and dioxygenated products. P450 2A13, 1B1, 1A2, 2C9, and 3A4 formed 1,2-epoxyacenaphthene at rates of 0.18, 5.3 2.4, 0.16, and 3.8 nmol/min/nmol P450, respectively. 1-Acenaphthenol, which induced Type I binding spectra with P450 2A13, was further oxidized by P450 2A13 but not P450 2A6. 1,2-Epoxyacenaphthene induced Type I binding spectra with P450 2A6 and 2A13 (Ks 1.8 and 0.16 μM, respectively) and was also oxidized to several oxidation products by these P450s. Molecular docking analysis suggested different orientations of acenaphthene, acenaphthylene, 1-acenaphthenol, and 1,2-epoxyacenaphthene in their interactions with P450 2A6 and 2A13. Neither of these four PAHs induced umu gene expression in a Salmonella typhimurium NM tester strain. These results suggest, for the first time, that acenaphthene and acenaphthylene are oxidized by human P450s 2A6 and 2A13 and other P450s to form several mono- and dioxygenated products. The results are of use in considering the biological and toxicological significance of these environmental PAHs in humans.
    Chemical Research in Toxicology 02/2015; 28(2). DOI:10.1021/tx500505y · 3.53 Impact Factor
  • Andrej Lajovic · Leslie D Nagy · F Peter Guengerich · Urban Bren
    [Show abstract] [Hide abstract]
    ABSTRACT: The carcinogenesis of urethane (ethyl carbamate), a by-product of fermentation that is consistently found in various food products, was investigated with a combination of kinetic experiments and quantum chemical calculations. The main objective of the study was to find ΔG(‡), the activation free energy for the rate-limiting step of the SN2 reaction between the ultimate carcinogen of urethane, vinyl carbamate epoxide (VCE), and different nucleobases of the DNA. In the experimental part, the second-order reaction rate constants for the formation of the main 7-(2-oxoethyl)guanine adduct in aqueous solutions of deoxyguanosine and in DNA were determined. A series of ab initio, density functional theory (DFT) and semiempirical molecular orbital (MO) calculations was then performed to determine the activation barriers for the reaction between VCE and nucleobases methylguanine, methyladenine, and methylcytosine. Effects of hydration were incorporated with the use of the solvent reaction field method of Tomasi and co-workers and the Langevine dipoles model of Florian and Warshel. The computational results for the main adduct were found to be in good agreement with the experiment, thus presenting a strong evidence for the validity of the proposed SN2 mechanism. This allowed us to predict the activation barriers of reactions leading to side products for which kinetic experiments have not yet been performed. Our calculations have shown that the main 7-(2-oxoethyl)deoxyguanosine adduct indeed forms preferentially because the emergence of other adducts either proceeds across a significantly higher activation barrier or the geometry of the reaction requires the Watson-Crick pairs of the DNA to be broken. The computational study also considered the questions of stereoselectivity, the ease of the elimination of the leaving group, and the relative contributions of the two possible reaction paths for the formation of the 1,N(2)-ethenoguanosine adduct.
    Chemical Research in Toxicology 02/2015; 28(4). DOI:10.1021/tx500459t · 3.53 Impact Factor
  • Source
    Slobodan Petar Rendic · F Peter Guengerich
    [Show abstract] [Hide abstract]
    ABSTRACT: Analyzing the literature resources used in our previous reports, we calculated the fractions of the oxidoreductase enzymes FMO (microsomal flavin-containing monooxygenase), AKR (aldo-keto reductase), MAO (monoamine oxidase), and cytochrome P450 participating in metabolic reactions. The calculations show that the fractions of P450s involved in metabolism of all chemicals (general chemicals, natural and physiological compounds, and drugs) are rather consistent in the findings that > 90% of enzymatic reactions are catalyzed by P450s. Regarding drug metabolism, three-fourths of the human P450 reactions can be accounted for by a set of five P450s: 1A2, 2C9, 2C19, 2D6, and 3A4, and the largest fraction of the P450 reactions is catalyzed by P450 3A enzymes. P450 3A4 participation in metabolic reactions of drugs varied from 13% for general chemicals to 27% for drugs.
    Chemical Research in Toxicology 01/2015; 28(1):38-42. DOI:10.1021/tx500444e · 3.53 Impact Factor
  • Source
    Slobodan Petar Rendic · F. P. Guengerich
  • Slobodan Petar Rendic · F.P. Guengerich
    [Show abstract] [Hide abstract]
    ABSTRACT: Analyzing the literature resources used in our previous reports, we calculated participation and the fractions of the oxidoreductase enzymes FMO (flavin containing monooxygenase), AKR (aldo-keto reductase), MAO (monoamine oxidase), and cytochrome P450 participating in metabolic reactions. The fractions of P450s involved in metabolism of drugs are rather consistent in the findings that ~75% of enzymatic reactions with drugs are catalyzed by P450s, ~90% of the P450 reactions can be accounted for by a set of five P450s: 1A2, 2C9, 2C19, 2D6, and 3A4, and the largest fraction of the P450 reactions is catalyzed by P450 3A enzymes.
    Chemical Research in Toxicology 01/2015; 28:38-42. · 3.53 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Cytochrome P450 (P450) 17A enzymes play a critical role in the oxidation of the steroids progesterone (Prog) and pregnenolone (Preg) to glucocorticoids and androgens. In mammals a single enzyme, P450 17A1, catalyzes both 17α-hydroxylation and a subsequent 17α,20-lyase reaction with both Prog and Preg. Teleost fish contain two 17A P450s: zebrafish P450 17A1 catalyzes both 17α-hydroxylation and lyase reactions with Prog and Preg, and P450 17A2 is more efficient in pregnenolone 17α-hydroxylation but does not catalyze the lyase reaction, even in the presence of cytochrome b5. P450 17A2 binds all substrates and products, although more loosely than P450 17A1. Pulse-chase and kinetic spectral experiments and modeling established that the 2-step P450 17A1 Prog oxidation is more distributive than the Preg reaction, i.e. 17α-OH product dissociates more prior to the lyase step. The drug orteronel selectively blocked the lyase reaction of P450 17A1, but only in the case of Prog. X-ray crystal structures of zebrafish P450 17A1 and 17A2 were obtained with the ligand abiraterone and, for P450 17A2, with Prog. Comparison of the two fish P450 17A-abiraterone structures with human P450 17A1 (DeVore, N. M., and Scott, E. E. (2013) Nature 482, 116-119) showed only a few differences near the active site, despite only ~50% identity among the three proteins. The P450 17A2 structure differed in four residues near the heme periphery. These residues may allow the proposed alternate ferric peroxide mechanism for the lyase reaction, or residues removed from the active site may allow conformations that lead to the lyase activity. Copyright © 2014, The American Society for Biochemistry and Molecular Biology.
    Journal of Biological Chemistry 12/2014; 290(6). DOI:10.1074/jbc.M114.627265 · 4.57 Impact Factor

Publication Stats

52k Citations
3,918.92 Total Impact Points


  • 1977–2015
    • Vanderbilt University
      • • Center in Molecular Toxicology
      • • Department of Biochemistry
      • • Department of Medicine
      Нашвилл, Michigan, United States
  • 2007–2008
    • Showa Pharmaceutical University
      Machida, Tōkyō, Japan
  • 2003
    • American University Washington D.C.
      Washington, Washington, D.C., United States
    • Hokkaido University
      • Graduate School of Pharmaceutical Sciences
      Sapporo-shi, Hokkaido, Japan
  • 2002
    • University of Queensland 
      • School of Biomedical Sciences
      Brisbane, Queensland, Australia
  • 2001
    • Université de Rennes 1
      Roazhon, Brittany, France
    • U.S. Food and Drug Administration
      Washington, Washington, D.C., United States
    • Technische Universität Kaiserslautern
      • Department of Food Chemistry and Environmental Toxicology
      Kaiserlautern, Rheinland-Pfalz, Germany
  • 1992–2001
    • Osaka Prefectural Institute of Public Health
      Ōsaka, Ōsaka, Japan
    • Hannover Medical School
      • Institute of Pharmacology
      Hanover, Lower Saxony, Germany
    • Johns Hopkins University
      • Department of Environmental Health Sciences
      Baltimore, Maryland, United States
    • Rutgers, The State University of New Jersey
      • Department of Chemical Biology
      Нью-Брансуик, New Jersey, United States
  • 1999
    • American Society for Pharmacology and Experimental Therapeutics
      Conshohocken, Pennsylvania, United States
  • 1998
    • National Taiwan University
      • College of Medicine
      Taipei, Taipei, Taiwan
  • 1997
    • Georg-August-Universität Göttingen
      Göttingen, Lower Saxony, Germany
  • 1996
    • Státní Zdravotní Ústav
      Praha, Praha, Czech Republic
  • 1995
    • University of Guelph
      • Department of Chemistry
      XIA, Ontario, Canada
    • French National Centre for Scientific Research
      • Centre de Recherche de Biochimie Macromoléculaire
      Lutetia Parisorum, Île-de-France, France
    • Pai Chai University
      Sŏul, Seoul, South Korea
  • 1991–1993
    • University of Cincinnati
      • • Center for Environmental Genetics
      • • Department of Environmental Health
      Cincinnati, Ohio, United States
    • Keio University
      Edo, Tōkyō, Japan
  • 1987–1992
    • University of Maryland, Baltimore
      • Division of Gastroenterology and Hepatology
      Baltimore, Maryland, United States
    • Westmead Hospital
      • Department of Medicine
      Sydney, New South Wales, Australia
  • 1990
    • University of Illinois, Urbana-Champaign
      Urbana, Illinois, United States
    • University of Texas Southwestern Medical Center
      • Department of Molecular Genetics
      Dallas, TX, United States
  • 1981–1990
    • University of Iowa
      • Department of Pharmacology
      Iowa City, IA, United States
  • 1989
    • The University of Arizona
      • Department of Pharmacology and Toxicology
      Tucson, Arizona, United States
  • 1988
    • Rutgers New Jersey Medical School
      • Department of Biochemistry and Molecular Biology (RWJ Medical School)
      Newark, New Jersey, United States
    • Harvard Medical School
      • Department of Biological Chemistry and Molecular Pharmacology
      Boston, Massachusetts, United States
  • 1984
    • National Cancer Institute (USA)
      Maryland, United States
  • 1982
    • Moncrief Cancer Institute
      Fort Worth, Texas, United States