42583-46-0Relevant articles and documents
Structural perturbations induced by the α-anomer of the aflatoxin B1 formamidopyrimidine adduct in duplex and single-strand DNA
Brown, Kyle L.,Voehler, Markus W.,Magee, Shane M.,Harris, Constance M.,Harris, Thomas M.,Stone, Michael P.
, p. 16096 - 16107 (2009)
The guanine N7 adduct of aflatoxin B1 exo-8,9-epoxide hydrolyzes to form the formamidopyrimidine (AFB-FAPY) adduct, which interconverts between R and β anomers. The β anomer is highly mutagenic in Escherichia coli, producing G → T transversions; it thermally stabilizes the DNA duplex. The AFB-α-FAPY adduct blocks replication; it destabilizes the DNA duplex. Herein, the structure of the AFB-α-FAPY adduct has been elucidated in 5′-d(C1T2A3T4X 5A6T7T8C9A 10)-3′ ?5′-d(T11G12A 13A14T15C16A17T 18A19G20)-3′ (X = AFB-α-FAPY) using molecular dynamics calculations restrained by NMR-derived distances and torsion angles. The AFB moiety intercalates on the 5′ face of the pyrimidine moiety at the damaged nucleotide between base pairs T4 ?A 17 and X5 ?C16, placing the FAPY C5-N 5 bond in the Ra axial conformation. Large perturbations of the ε and ζ backbone torsion angles are observed, and the base stacking register of the duplex is perturbed. The deoxyribose orientation shifts to become parallel to the FAPY base and displaced toward the minor groove. Intrastrand stacking between the AFB moiety and the 5′ neighbor thymine remains, but strong interstrand stacking is not observed. A hydrogen bond between the formyl group and the exocyclic amine of the 3′-neighbor adenine stabilizes the E conformation of the formamide moiety. NMR studies reveal a similar 5′-intercalation of the AFB moiety for the AFB-β-FAPY adduct in the tetramer 5′-d(C1T 2X3A4)-3′, involving the Ra axial conformation of the FAPY C5-N5 bond and the E conformation of the formamide moiety. Since in duplex DNA the AFB moiety of the AFB-β-FAPY adduct also intercalates on the 5′ side of the pyrimidine moiety at the damaged nucleotide, we conclude that favorable 5′-stacking leads to the Ra conformational preference about the C5-N5 bond; the same conformational preference about this bond is also observed at the nucleoside and base levels. The structural distortions and the less favorable stacking interactions induced by the AFB-α-FAPY adduct explain its lower stability as compared to the AFB-β-FAPY adduct in duplex DNA. In this DNA sequence, hydrogen bonding between the formyl oxygen and the exocyclic amine of the 3′-neighboring adenine stabilizing the E configuration of the formamide moiety is also observed for the AFB-β-FAPY adduct, and suggests that the identity of the 3′-neighbor nucleotide modulates the stability and biological processing of AFB adducts.
Preparation and Characterization of an Aflatoxin B1 Adduct with the Oligodeoxynucleotide d(ATCGAT)2
Gopalakrishnan, S.,Stone, Michael P.,Harris, Thomas M.
, p. 7232 - 7239 (1989)
Preparation of a double-stranded aflatoxin B1-oligodeoxynucleotide adduct by direct addition of aflatoxin B1 8,9-epoxide to d(ATCGAT)2 is described.Reaction occurred rapidly at 5 deg C to give a high yield of adduct.The reaction reached a limiting stoichiometry of 1:1 aflatoxin B1 to d(ATCGAT)2.The major product, which exhibited UV absorbance at 360 nm, was identified as 8,9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B1*d(ATCGAT).Reversed-phase HPLC yielded equimolar quantities of unmodified d(ATCGAT) and 8,9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B1.Acid hydrolysis followed by reverse-phase HPLC yielded 8,9-dihydro-8-N7-guanyl-9-hydroxyaflatoxin B1 .Gentle heating at pH 8 and subsequent acid hydrolysis gave products consistent with formation of formamidopyrimidine (FAPY) aflatoxin B1 derivatives.Circular dichroism measurements showed negative ellipticity at 360 nm and an increase in positive ellipticity at 260 nm as compared to unmodified d(ATCGAT)2.UV melting curves demonstrated that adduct formation increased duplex stability.Spontaneous depurination of the modified duplex was observed but was sufficiently slow at 5 deg C and neutral pH to obtain NMR spectra. 1H NMR spectra exhibited a doubling of oligodeoxynucleotide resonances upon adduct formation due to loss of strand symmetry; strand exchange between modified and unmodified oligodeoxynucleotide duplexes was slow on the NMR time scale.Adduct formation resulted in increased shielding for aflatoxin protons.The C8 proton of the modified guanine was not observed in the 1H NMR spectrum in D2O, but an additional signal tentatively assigned as that proton was observed at 9.75 ppm in the spectrum in H2O.This signal was not observed after mild basic hydrolysis of the duplex cationic adduct.Six hydrogen-bonded NH resonances were observed between 12 and 14 ppm. 31P NMR showed a doubling of resonances and one signal shifted downfield at least 1 ppm.A structure for the adduct is proposed in which the aflatoxin moiety is intercalated above the 5' face of the modified guanine.
Influence of P450 3A4 SRS-2 residues on cooperativity and/or regioselectivity of aflatoxin B1 oxidation
Xue, Linlong,Wang, Huifen Faye,Wang, Qinmi,Szklarz, Grazyna D.,Domanski, Tammy L.,Halpert, James R.,Correia, Maria Almira
, p. 483 - 491 (2007/10/03)
The major human liver drug-metabolizing cytochrome P450 enzymes P450 3A4 and P450 3A5 share >85% amino acid sequence identity yet exhibit different regioselectivity toward aflatoxin B1 (AFB1) biotransformation [Gillam et al. (1995) Arch. Biochem. Biophys. 317, 74-384]. P450 3A4 prefers AFB1 3α-hydroxylation, which detoxifies and subsequently eliminates the hepatotoxin, over AFB1 exo-8,9-oxidation. P450 3A5, on the other hand, is a relatively sluggish 3α-hydroxylase and converts AFB1 predominantly to the genotoxic exo-8,9-epoxide. Using a combination of approaches (sequence alignment, homology modeling and site-directed mutagenesis), we have previously identified several divergent residues in four of the six putative substrate recognition sites (SRSs) of P450 3A4, which when replaced individually with the corresponding amino acid of P450 3A5, resulted in a significant switch of the characteristic P450 3A4 AFB1 regioselectivity toward that of P450 3A5 [Wang et al. (1998) Biochemistry 37, 12536-12545]. In particular, residues N206 and L210 in SRS-2 were found to be critical for AFB1 detoxification via 3α-hydroxylation, and the corresponding mutants N206S and L210F most closely mimicked P450 3A5, not only in its regioselectivity of AFB1 metabolism but also in its overall functional capacity. We have now further explored the plausible reasons for such relative inactivity of the SRS-2 mutants by examining N206S and additional mutants (L210A, L211F, L211A, and N206E) and found that the dramatically lowered activities of the N206S mutant are accompanied by a loss of cooperativity of AFB1 oxidation. Molecular dynamics analyses with an existing P450 3A4 homology model [Szklarz and Halpert (1997) J. Comput. Aided Mol. Des. 11,265] suggested that N206 (helix F) interacts with E244 (helix G), creating a salt bridge that stabilizes the protein structure and/or defines the active site cavity. To examine this possibility, several E244 mutants (E244A, V, N, S) were tested, of which E244S was the most notable for its relatively greater impairment of P450 3A4-dependent AFB1 3α-hydroxylation. However, the results with these E244 mutants failed to validate the N206-E244 interaction predicted from these molecular dynamics analyses. Collectively, our findings to date have led us to reconsider our original interpretations and to reexamine them in the light of AFB1 molecular modeling analyses with a newly refined P450 3A4 homology model. These analyses predicted that F304 in SRS-4 (I-helix) plays a pivotal role in AFB1 binding at the active site in either orientation leading to 3α- or exo-8,9-oxidation. Consistent with this prediction, conversion of F304 to Ala abolished P450 3A4-dependent AFB1 3α-hydroxylation and exo-8,9-oxidation.
Interaction of aflatoxin B1 with cytochrome P450 2A5 and its mutants: correlation with metabolic activation and toxicity.
Pelkonen,Lang,Negishi,Wild,Juvonen
, p. 85 - 90 (2007/10/03)
Among members of the mouse cytochrome P450 2A family, P450 2A5 is the best catalyst of aflatoxin B1 (AFB1) oxidation to its 8,9-epoxide (Pelkonen, P., Lang, M., Wild, C. P., Negishi, M., and Juvonen, R. O. (1994) Eur. J. Pharmacol., Environ. Toxicol. Pharmacol. Sect. 292, 67-73). Here we studied the role of amino acid residues 209 and 365 of the P450 2A5 in the metabolism and toxicity of AFB1 using recombinant yeasts. The two sites have previously been shown to be essential in the interaction of coumarin and steroids with the P450 2A5. Reducing the size of the amino acid at position 209 or introducing a negatively charged residue at this site increased the 8,9-epoxidation of AFB1 compared to the wild type. In addition, replacing the hydrophobic amino acid at the 365 position with a positively charged lysine residue strongly decreased the metabolism of AFB1. These mutations changed the KM values generally less than the Vmax values. The changes in AFB1 metabolism contrast with the changes in coumarin 7-hydroxylation caused by these amino acid substitutions, since reducing the size of the 209 residue strongly reduced coumarin metabolism and increased the K(M) values. On the other hand, the results with AFB1 are similar to those obtained with steroid hydroxylation. This suggests that the size of the substrate is important when interacting with the residue 209 of the protein. The catalytic parameters of AFB1 correlated generally with its toxicity to the recombinant yeasts expressing the activating enzyme and with the binding of AFB1 to yeast DNA. Furthermore high affinity substrates and inhibitors (e.g., methoxsalen, metyrapone, coumarin 311, 7-methylcoumarin, coumarin, and pilocarpine) of P450 2A5 could efficiently block the toxicity of AFB1. It is suggested that the recombinant yeasts expressing engineered P450 enzymes are a useful model to understand the substrate protein interactions, to study the relationship of metabolic parameters to toxicity, and to test potential inhibitors of metabolism based toxicity.
