Two arylamine N-acetyltransferases from chicken pineal gland as identified by cDNA cloning

A cDNA library prepared from the poly(A)-rich RNA of the chicken pineal gland obtained at night was screened with the 32P-labeled cDNA of arylamine N-acetyltransferase from the chicken liver recently isolated in this laboratory. Two positive clones (p-NAT-3 and p-NAT-10) that cross-hybridized with the liver cDNA were isolated. The cDNAs did not cross-hybridize each other under a high stringency, indicating that they corresponded to different mRNAs. When the cDNAs were inserted into an expression vector pcDL1 under the control of the early promoter of simian virus 40 and introduced into Chinese hamster ovary cells, both cDNAs expressed arylamine N-acetyltransferase activity in the transfected cells. The nucleotide sequences of the cDNAs were determined, from which amino acid sequences were deduced. Both cDNAs coded for 290 amino acids. Similarities in amino acid sequences were about 60% between p-NAT-3, p-NAT-10 and liver N-acetyltransferases. Poly(A)-rich RNA blot hybridization analysis indicated that p-NAT-3 cDNA detected a 2.2-kb band with the poly(A)-rich RNAs from the brain, gut and, less intensively, spleen, liver and kidney, while p-NAT-10 cDNA hybridized only with the poly(A)-rich RNA from the kidney. Neither cDNA detected any hybridization band with the poly(A)-rich RNA from the pineal gland, suggesting that the contents were low. Genomic Southern blot hybridization analysis showed that p-NAT-3, p-NAT-10 and liver N-acetyltransferases were encoded in a separate single gene. The properties of the enzymes expressed in the transfected cells were compared with N-acetyltransferases from the pineal gland, brain and kidney. On a DEAE-cellulose column, the kidney and p-NAT-10 enzymes appeared in the effluent fraction, whereas the brain and p-NAT-3 enzymes were eluted from the column with a gradient elution at 0.08 M NaCl. The supernatant of the pineal gland obtained in the daytime showed two peaks appearing in the effluent fraction and the eluate fraction at 0.08 M NaCl. The substrate specificity of these enzymes were examined with p-phenetidine, 2-aminofluorene, tryptamine and phenylethylamine as substrates. All the enzymes preferred arylamines to arylalkylamines, indicating that both p-NAT-3 and p-NAT-10 cDNAs encoded arylamine N-acetyltransferases.     

Heterocyclic aromatic amine content of selected beef flavors

Previous work has shown that meat extracts contain potent mutagenic and/or carcinogenic heterocyclic aromatic amines (HAAs). Because meat extracts and some beef flavors are produced from similar precursors and processing steps, the beef flavors may also contain HAAs. This study analyzed 24 commercial beef flavors and 2 food-grade beef extracts for creatine and creatinine concentrations, mutagenic activity and HAA concentrations (IQ, MeIQ, MeIQ_x, DiMeIQ_x, Glu-P-1, Glu-P-2 and PhIP). The creatine and creatinine levels of the flavors ranged from 0 to 73 and from 0 to 21 mg/g (dry wt.), respectively. The mutagenic activities of the flavors ranged from 0 to 3200 Salmonella typhimurium TA98 revertants/g (dry wt.). No direct relationship was found between creatine and/or creatinine concentrations and mutagenic activities. However, flavors with high creatine (> 1.5 mg/g) or creatinine (> 2 mg/g) levels exhibited higher mutagenic activities than did flavors with low levels of these compounds. Flavors with high mutagenic activities (> 1500 revertants/g) contained measurable amounts of HAAs. Three flavors contained MeIQ_x (7.2–21.2 ng/g [dry wt.]) and one contained DiMeIQ_x (4.2 ng/g [dry wt.]).     

Crystallization and properties of aromatic amine dehydrogenase from Pseudomonas sp

An amine dehydrogenase was purified to homogeneity from an extract of a bacterium of the genus Pseudomonas grown in a medium containing beta-phenylethylamine as a sole carbon source and obtained in a crystalline form with about 100-fold purification. The purified enzyme catalyzed the oxidative deamination of various aromatic amines as well as some aliphatic amines to a lesser extent. An artificial electron acceptor such as phenazine methosulfate was required for the catalysis. The molecular weight determined by sedimentation equilibrium was 103,000 and the molecule seemed to be composed of two pairs of two nonidentical subunits (Mr 46,000 and 8000). The enzyme had a dull yellow-green color with an absorption maximum at 445 nm and this chromophore appeared to be involved in the catalytic action of the enzyme.     

Purification and characterization of aromatic amine dehydrogenase from Alcaligenes xylosoxidans

Aromatic amine dehydrogenase was purified and characterized from Alcaligenes xylosoxidans IFO13495 grown on beta-phenylethylamine. The molecular mass of the enzyme was 95.5 kDa. The enzyme consisted of heterotetrameric subunits (alpha2beta2) with two different molecular masses of 42.3 kDa and 15.2 kDa. The N-terminal amino acid sequences of the alpha-subunit (42.3-kDa subunit) and the beta-subunit (15.2-kDa subunit) were DLPIEELXGGTRLPP and APAAGNKXPQMDDTA respectively. The enzyme had a quinone cofactor in the beta-subunit and showed a typical absorption spectrum of tryptophan tryptophylquinone-containing quinoprotein showing maxima at 435 nm in the oxidized form and 330 nm in the reduced form. The pH optima of the enzyme activity for histamine, tyramine, and beta-phenylethylamine were the same at 8.0. The enzyme retained full activity after incubation at 70 degrees C for 40 min. It readily oxidized various aromatic amines as well as some aliphatic amines. The Michaelis constants for phenazine methosulfate, beta-phenylethylamine, tyramine, and histamine were 48.1, 1.8, 6.9, and 171 microM respectively. The enzyme activity was strongly inhibited by carbonyl reagents. The enzyme could be stored without appreciable loss of enzyme activity at 4 degrees C for one month at least in phosphate buffer (pH 7.0).     

Atomic level insight into the oxidative half-reaction of aromatic amine dehydrogenase

The quinoprotein aromatic amine dehydrogenase (AADH) uses a covalently bound tryptophan tryptophylquinone (TTQ) cofactor to oxidatively deaminate primary aromatic amines. Recent crystal structures have provided insight into the reductive half-reaction. In contrast, no atomic details are available for the oxidative half-reaction. The TTQ O7 hydroxyl group is protonated during reduction, but it is unclear how this proton can be removed during the oxidative half-reaction. Furthermore, compared with the electron transfer from the N-quinol form, electron transfer from the non-physiological O-quinol form to azurin is significantly slower. Here we report crystal structures of the O-quinol, N-quinol, and N-semiquinone forms of AADH. A comparison of oxidized and substrate reduced AADH species reveals changes in the TTQ-containing subunit, extending from residues in the immediate vicinity of the N-quinol to the putative azurin docking site, suggesting a mechanism whereby TTQ redox state influences interprotein electron transfer. In contrast, chemical reduction of the TTQ center has no significant effect on protein conformation. Furthermore, structural reorganization upon substrate reduction places a water molecule near TTQ O7 where it can act as proton acceptor. The structure of the N-semiquinone, however, is essentially similar to oxidized AADH. Surprisingly, in the presence of substrate a covalent N-semiquinone substrate adduct is observed. To our knowledge this is the first detailed insight into a complex, branching mechanism of quinone oxidation where significant structural reorganization upon reduction of the quinone center directly influences formation of the electron transfer complex and nature of the electron transfer process.     

Reduction of aromatic steroidal A rings by lithium in ethyl amine.

The reduction of 3-methoxy-estra-1,3,5(10)-trien-17beta-ol (6) and 13-ethyl-3-ethoxy-gona-1,3,5(10)-triene-11alpha,17beta-diol (2) by lithium in ethyl amine in the absence of a proton source is described. Both reductions, contrary to the reports of previous investigators, which indicated the 4-ene to be the main reaction product, gave a complex mixture of products. In the case of the reduction of 2, which is an intermediate in the synthesis of the progestagen desogestrel (1), we obtained the expected known 13-ethyl-gona-4-ene-11alpha,17beta-diol (4) in small amounts and three new steroidal monoenes, 13-ethyl-gona-5(10)-ene-11alpha,17beta-diol (11), 13-ethyl-gona-5(6)-ene-11alpha,17beta-diol (12), and 13-ethyl-gona-1(10)-ene-11alpha,17beta-diol (13). These compounds were characterized as the 11,17-diacetates with the 5(10)-ene 11 being the major compound.     

The aromatic amine carcinogens o-toluidine and o-anisidine induce free radicals and intrachromosomal recombination in Saccharomyces cerevisiae

Aniline-based aromatic amine carcinogens are poorly detected in short-term mutagenicity assays such as the Salmonella reverse mutation (Ames) assay. More information on the mechanism of toxicity of such Salmonella-negative carcinogens is needed. Aniline and o-toluidine are negative in the Ames assay, but induce deletions (DEL) due to intrachromosomal recombination in Saccharomyces cerevisiae with an apparent threshold. We show here that the DEL assay also detects the genotoxic activity of another aromatic amine carcinogen, o-anisidine, which is also negative in the Salmonella assay. We also show that the DEL assay distinguishes between o-anisidine and its non-carcinogenic structural analog 2, 4-dimethoxyaniline. We have investigated whether the ability of the DEL assay to detect the carcinogens and to distinguish between the carcinogen/non-carcinogen pair is linked to rises in intracellular free radical species following exposure to the carcinogens. Toxicity induced by all three compounds was reduced in the presence of the free radical scavenger and antioxidant N-acetyl cysteine, recombination induced by o-anisidine and o-toluidine was also reduced by N-acetyl cysteine. All three compounds induced oxidation of the free radical-sensitive reporter compound dichlorofluorescin diacetate. Superoxide dismutase-deficient strains, however, were hypersensitive to cytotoxicity induced by o-toluidine and o-anisidine but not by the non-carcinogen 2,4-dimethoxyaniline, indicating a different potential for generating superoxide radical between the carcinogens and the non-carcinogen analog. The results indicate that the yeast DEL assay is a useful tool for investigating the genotoxic activity of aromatic amine carcinogens.     

Mechanism of aromatic amine carcinogen bypass by the Y-family polymerase,Dpo4

Bulky DNA damage inhibits DNA synthesis by replicative polymerases and often requires the action of error prone bypass polymerases. The exact mechanism governing adduct-induced mutagenesis and its dependence on the DNA sequence context remains unclear. In this work, we characterize Dpo4 binding conformations and activity with DNA templates modified with the carcinogenic DNA adducts, 2-aminofluoene (AF) or N-acetyl-2-aminofluorene (AAF), using single-molecule FRET (smFRET) analysis and DNA synthesis extension assays. We find that in the absence of dNTPs, both adducts alter polymerase binding as measured by smFRET, but the addition of dNTPs induces the formation of a ternary complex having what appears to be a conformation similar to the one observed with an unmodified DNA template. We also observe that the misincorporation pathways for each adduct present significant differences: while an AF adduct induces a structure consistent with the previously observed primer-template looped structure, its acetylated counterpart uses a different mechanism, one consistent with a dNTP-stabilized misalignment mechanism.     

The avian pineal gland

The pineal gland plays a key role in the control of the daily and seasonal rhythms in most vertebrate species. In mammals, rhythmic melatonin (MT) release from the pineal gland is controlled by the suprachiasmatic nucleus via the sympathetic nervous system. In most non-mammalian species, including birds, the pineal gland contains a self-sustained circadian oscillator and several input channels to synchronize the clock, including direct light sensitivity. Avian pineal glands maintain rhythmic activity for days under in vitro conditions. Several physical (light, temperature, and magnetic field) and biochemical (Vasoactive intestinal polypeptide (VIP), norepinephrine, PACAP, etc.) input channels, influencing release of melatonin are also functional in vitro, rendering the explanted avian pineal an excellent model to study the circadian biological clock. Using a perifusion system, we here report that the phase of the circadian melatonin rhythm of the explanted chicken pineal gland can be entrained easily to photoperiods whose length approximates 24 h, even if the light period is extremely short, i.e., 3L:21D. When the length of the photoperiod significantly differs from 24 h, the endogenous MT rhythm becomes distorted and does not follow the light-dark cycle. When explanted chicken pineal fragments were exposed to various drugs targeting specific components of intracellular signal transduction cascades, only those affecting the cAMP-protein kinase-A system modified the MT release temporarily without phase-shifting the rhythm in MT release. The potential role of cGMP remains to be investigated.     

Gated and ungated electron transfer reactions from aromatic amine dehydrogenase to azurin.

Interprotein electron transfer (ET) occurs between the tryptophan tryptophylquinone (TTQ) prosthetic group of aromatic amine dehydrogenase (AADH) and copper of azurin. The ET reactions from two chemically distinct reduced forms of TTQ were studied: an O-quinol form that was generated by reduction by dithionite, and an N-quinol form that was generated by reduction by substrate. It was previously shown that on reduction by substrate, an amino group displaces a carbonyl oxygen on TTQ, and that this significantly alters the rate of its oxidation by azurin (Hyun, Y-L., and Davidson V. L. (1995) Biochemistry 34, 12249-12254). To determine the basis for this change in reactivity, comparative kinetic and thermodynamic analyses of the ET reactions from the O-quinol and N-quinol forms of TTQ in AADH to the copper of azurin were performed. The reaction of the O-quinol exhibited values of electronic coupling (H(AB)) of 0.13 cm(-1) and reorganizational energy (lambda) of 1.6 eV, and predicted an ET distance of approximately 15 A. These results are consistent with the ET event being the rate-determining step for the redox reaction. Analysis of the reaction of the N-quinol by Marcus theory yielded an H(AB) which exceeded the nonadiabatic limit and predicted a negative ET distance. These results are diagnostic of a gated ET reaction. Solvent deuterium kinetic isotope effects of 1.5 and 3.2 were obtained, respectively, for the ET reactions from O-quinol and N-quinol AADH indicating that transfer of an exchangeable proton was involved in the rate-limiting reaction step which gates ET from the N-quinol, but not the O-quinol. These results are compared with those for the ET reactions from another TTQ enzyme, methylamine dehydrogenase, to amicyanin. The mechanism by which the ET reaction of the N-quinol is gated is also related to mechanisms of other gated interprotein ET reactions.     

Observing translesion synthesis of an aromatic amine DNA adduct by a high-fidelity DNA polymerase

Aromatic amines have been studied for more than a half-century as model carcinogens representing a class of chemicals that form bulky adducts to the C8 position of guanine in DNA. Among these guanine adducts, the N-(2'-deoxyguanosin-8-yl)-aminofluorene (G-AF) and N-2-(2'-deoxyguanosin-8-yl)-acetylaminofluorene (G-AAF) derivatives are the best studied. Although G-AF and G-AAF differ by only an acetyl group, they exert different effects on DNA replication by replicative and high-fidelity DNA polymerases. Translesion synthesis of G-AF is achieved with high-fidelity polymerases, whereas replication of G-AAF requires specialized bypass polymerases. Here we have presented structures of G-AF as it undergoes one round of accurate replication by a high-fidelity DNA polymerase. Nucleotide incorporation opposite G-AF is achieved in solution and in the crystal, revealing how the polymerase accommodates and replicates past G-AF, but not G-AAF. Like an unmodified guanine, G-AF adopts a conformation that allows it to form Watson-Crick hydrogen bonds with an opposing cytosine that results in protrusion of the bulky fluorene moiety into the major groove. Although incorporation opposite G-AF is observed, the C:G-AF base pair induces distortions to the polymerase active site that slow translesion synthesis.     

Hierarchy of structures in the family of amine templated open-framework gallium arsenates

Five new gallium arsenate compounds [C₂N₂H₁₀][Ga(H₂AsO₄)(HAsO₄)₂]·H₂O, I; [C₂N₂H₁₀][Ga(OH)(AsO₄)]₂, II; [C₂N₂H₁₀][GaF(AsO₄)]₂, III; [C₃N₂H₁₂][Ga(OH)(AsO₄)]₂, IV; [Ga₂F₃(AsO₄)(HAsO₄)]·2H₃O, V, have been synthesized under hydrothermal conditions and the structures determined employing single crystal X-ray diffraction studies. All the structures consist of octahedral gallium and tetrahedral arsenate units connected together forming a hierarchy of structures. Thus, one- (I), two- (II and IV) and three-dimensionally (III and V) extended structures have been observed. The Ga-O(H)/F-Ga connectivity in some of the structures suggests the coordination requirements posed by the octahedral gallium in these compounds. The observation of only one type of secondary building unit in the structures of III (SBU-4) and V (spiro-5) is unique and noteworthy. All the compounds have been characterized by a variety of techniques that include powder XRD, IR, and TGA.     

Induction of genetic tandem duplications in Salmonella by polycyclic aromatic hydrocarbon and amine carcinogens

6 polycyclic aromatic hydrocarbon or similar amine carcinogens were tested as inducers of genetic tandem duplications in a rough strain of Salmonella typhimurium. When metabolically activated by rat-liver microsomes, all 6 were active in inducing genetic tandem duplications, yielding from over 3 times to almost 14 times as many tandem duplicants per viable bacterium as did concurrent uninduced control cultures. These results extend the number and chemical diversity of carcinogens shown to induce genetic duplications in bacterial tester systems. We suggest that polycyclic hydrocarbon carcinogens may act in carcinogenesis by inducing genetic duplications or other genetic rearrangements. Duplication induction may be a useful genetic endpoint for screening potential carcinogens.     

Crystal structure of mycothiol synthase (Rv0819) from Mycobacterium tuberculosis shows structural homology to the GNAT family of N-acetyltransferases

Mycothiol is the predominant low-molecular weight thiol produced by actinomycetes, including Mycobacterium tuberculosis. The last reaction in the biosynthetic pathway for mycothiol is catalyzed by mycothiol synthase (MshD), which acetylates the cysteinyl amine of cysteine-glucosamine-inositol (Cys-GlcN-Ins). The crystal structure of MshD was determined in the presence of coenzyme A and acetyl-CoA. MshD consists of two tandem-repeated domains, each exhibiting the Gcn5-related N-acetyltransferase (GNAT) fold. These two domains superimpose with a root-mean-square deviation of 1.7 A over 88 residues, and each was found to bind one molecule of coenzyme, although the binding sites are quite different. The C-terminal domain has a similar active site to many GNAT members in which the acetyl group of the coenzyme is presented to an open active site slot. However, acetyl-CoA bound to the N-terminal domain is buried, and is apparently not positioned to promote acetyl transfer. A modeled substrate complex indicates that Cys-GlcN-Ins would only fill a portion of a negatively charged channel located between the two domains. This is the first structure determined for an enzyme involved in the biosynthesis of mycothiol.     

The interaction of methanol, rat-liver S9 and the aromatic amine 2,4-diaminotoluene produces a new mutagenic compound

Methanol is a widely used solvent for organic compounds and a human toxicant. In our studies of the metabolism of aromatic amines in the Ames/Salmonella assay, we observed a rapid and quantitative conversion of the mutagenic and carcinogenic aromatic amine 2,4-diaminotoluene (2,4-DAT) to a single product. This product was only produced in the presence of methanol, and not other organic solvents. Isolation of this product showed that it was highly mutagenic in Salmonella TA98 with S9 activation. Characterization of the product of the interaction of methanol and 2,4-DAT indicated that methanol is activated to a reactive intermediate, probably formaldehyde, by the 9000 X g supernatant used in the Ames/Salmonella assay. The formaldehyde subsequently reacts with 2,4-DAT to form the mutagenic product, identified as bis-5,5'(2,4,2',4'-tetraaminotolyl)methane. Results of this study demonstrate that methanol may be an inappropriate solvent for mutation and metabolism studies of aromatic amines and possibly other chemicals, and that solvent-xenobiotic interactions may in some cases lead to the misinterpretation of results.