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Paul F. Fitzpatrick - One of the best experts on this subject based on the ideXlab platform.
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Nitroalkane oxidase: Structure and mechanism.
Archives of biochemistry and biophysics, 2017Co-Authors: Paul F. FitzpatrickAbstract:The flavoprotein Nitroalkane oxidase catalyzes the oxidation of neutral Nitroalkanes to the corresponding aldehydes or ketones, releasing nitrite and transferring electrons to O2 to form H2O2. A combination of solution and structural analyses have provided a detailed understanding of the mechanism of this enzyme.
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solvent isotope and viscosity effects on the steady state kinetics of the flavoprotein Nitroalkane oxidase
FEBS Letters, 2013Co-Authors: Giovanni Gadda, Paul F. FitzpatrickAbstract:The flavoprotein Nitroalkane oxidase catalyzes the oxidative denitrification of a broad range of primary and secondary Nitroalkanes to yield the respective aldehydes or ketones, hydrogen peroxide and nitrite. With nitroethane as substrate the D2O(kcat/KM) value is 0.6 and the D2Okcat value is 2.4. The kcat proton inventory is consistent with a single exchangeable proton in flight, while the kcat/KM is consistent with either a single proton in flight in the transition state or a medium effect. Increasing the solvent viscosity did not affect the kcat or kcat/KM value significantly, establishing that nitroethane binding is at equilibrium and that product release does not limit kcat.
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identification of a hypothetical protein from podospora anserina as a Nitroalkane oxidase
Biochemistry, 2010Co-Authors: Jose R Tormos, Colette S Daubner, Alexander B Taylor, John P Hart, Paul F. FitzpatrickAbstract:The flavoprotein Nitroalkane oxidase (NAO) from Fusarium oxysporum catalyzes the oxidation of primary and secondary Nitroalkanes to their respective aldehydes and ketones. Structurally, the enzyme is a member of the acyl-CoA dehydrogenase superfamily. To date no enzymes other than that from F. oxysporum have been annotated as NAOs. To identify additional potential NAOs, the available database was searched for enzymes in which the active site residues Asp402, Arg409, and Ser276 were conserved. Of the several fungal enzymes identified in this fashion, PODANSg2158 from Podospora anserina was selected for expression and characterization. The recombinant enzyme is a flavoprotein with activity on Nitroalkanes comparable to the F. oxysporum NAO, although the substrate specificity is somewhat different. Asp399, Arg406, and Ser273 in PODANSg2158 correspond to the active site triad in F. oxysporum NAO. The k(cat)/K(M)-pH profile with nitroethane shows a pK(a) of 5.9 that is assigned to Asp399 as the active site base. Mutation of Asp399 to asparagine decreases the k(cat)/K(M) value for nitroethane over 2 orders of magnitude. The R406K and S373A mutations decrease this kinetic parameter by 64- and 3-fold, respectively. The structure of PODANSg2158 has been determined at a resolution of 2.0 A, confirming its identification as an NAO.
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characterization of active site residues of Nitroalkane oxidase
Bioorganic Chemistry, 2010Co-Authors: Michael P. Valley, Nana S Fenny, Shah R Ali, Paul F. FitzpatrickAbstract:The flavoenzyme Nitroalkane oxidase catalyzes the oxidation of primary and secondary Nitroalkanes to the corresponding aldehydes and ketones plus nitrite. The structure of the enzyme shows that Ser171 forms a hydrogen bond to the flavin N5, suggesting that it plays a role in catalysis. Cys397 and Tyr398 were previously identified by chemical modification as potential active site residues. To more directly probe the roles of these residues, the S171A, S171V, S171T, C397S, and Y398F enzymes have been characterized with nitroethane as substrate. The C397S and Y398 enzymes were less stable than the wild-type enzyme, and the C397S enzyme routinely contained a substoichiometric amount of FAD. Analysis of the steady-state kinetic parameters for the mutant enzymes, including deuterium isotope effects, establishes that all of the mutations result in decreases in the rate constants for removal of the substrate proton by approximately 5-fold and decreases in the rate constant for product release of approximately 2-fold. Only the S171V and S171T mutations alter the rate constant for flavin oxidation. These results establish that these residues are not involved in catalysis, but rather are required for maintaining the protein structure.
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Crystal structures of intermediates in the Nitroalkane oxidase reaction.
Biochemistry, 2009Co-Authors: Annie Heroux, Paul F. Fitzpatrick, Dragana M. Bozinovski, Michael P. Valley, Allen M. OrvilleAbstract:The flavoenzyme Nitroalkane oxidase is a member of the acyl-CoA dehydrogenase superfamily. Nitroalkane oxidase catalyzes the oxidation of neutral Nitroalkanes to nitrite and the corresponding aldehydes or ketones. Crystal structures to 2.2 A resolution or better of enzyme complexes with bound substrates and of a trapped substrate-flavin adduct are described. The D402N enzyme has no detectable activity with neutral Nitroalkanes [Valley, M. P., and Fitzpatrick, P. F. (2003) J. Am. Chem. Soc. 125, 8738-8739]. The structure of the D402N enzyme crystallized in the presence of 1-nitrohexane or 1-nitrooctane shows the presence of the substrate in the binding site. The aliphatic chain of the substrate extends into a tunnel leading to the enzyme surface. The oxygens of the substrate nitro group interact both with amino acid residues and with the 2'-hydroxyl of the FAD. When Nitroalkane oxidase oxidizes Nitroalkanes in the presence of cyanide, an electrophilic flavin imine intermediate can be trapped [Valley, M. P., Tichy, S. E., and Fitzpatrick, P. F. (2005) J. Am. Chem. Soc. 127, 2062-2066]. The structure of the enzyme trapped with cyanide during oxidation of 1-nitrohexane shows the presence of the modified flavin. A continuous hydrogen bond network connects the nitrogen of the CN-hexyl-FAD through the FAD 2'-hydroxyl to a chain of water molecules extending to the protein surface. Together, our complementary approaches provide strong evidence that the flavin cofactor is in the appropriate oxidation state and correlates well with the putative intermediate state observed within each of the crystal structures. Consequently, these results provide important structural descriptions of several steps along the Nitroalkane oxidase reaction cycle.
Giovanni Gadda - One of the best experts on this subject based on the ideXlab platform.
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solvent isotope and viscosity effects on the steady state kinetics of the flavoprotein Nitroalkane oxidase
FEBS Letters, 2013Co-Authors: Giovanni Gadda, Paul F. FitzpatrickAbstract:The flavoprotein Nitroalkane oxidase catalyzes the oxidative denitrification of a broad range of primary and secondary Nitroalkanes to yield the respective aldehydes or ketones, hydrogen peroxide and nitrite. With nitroethane as substrate the D2O(kcat/KM) value is 0.6 and the D2Okcat value is 2.4. The kcat proton inventory is consistent with a single exchangeable proton in flight, while the kcat/KM is consistent with either a single proton in flight in the transition state or a medium effect. Increasing the solvent viscosity did not affect the kcat or kcat/KM value significantly, establishing that nitroethane binding is at equilibrium and that product release does not limit kcat.
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cloning of Nitroalkane oxidase from fusarium oxysporum identifies a new member of the acyl coa dehydrogenase superfamily
Proceedings of the National Academy of Sciences of the United States of America, 2002Co-Authors: Giovanni Gadda, Michael P. Valley, Colette S Daubner, Paul F. FitzpatrickAbstract:The flavoprotein Nitroalkane oxidase (NAO) from Fusarium oxysporum catalyzes the oxidation of Nitroalkanes to the respective aldehydes with production of nitrite and hydrogen peroxide. The sequences of several peptides from the fungal enzyme were used to design oligonucleotides for the isolation of a portion of the NAO gene from an F. oxysporum genomic DNA preparation. This sequence was used to clone the cDNA for NAO from an F. oxysporum cDNA library. The sequence of the cloned cDNA showed that NOA is a member of the acyl-CoA dehydrogenase (ACAD) superfamily. The members of this family share with NAO a mechanism that is initiated by proton removal from carbon, suggesting a common chemical reaction for this superfamily. NAO was expressed in Escherichia coli and the recombinant enzyme was characterized. Recombinant NAO has identical kinetic parameters to enzyme isolated from F. oxysporum but is isolated with oxidized FAD rather than the nitrobutyl-FAD found in the fungal enzyme. NAO purified from E. coli or from F. oxysporum has no detectable ACAD activity on short- or medium-chain acyl CoAs, and medium-chain acyl-CoA dehydrogenase and short-chain acyl-CoA dehydrogenase are unable to catalyze oxidation of Nitroalkanes.
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Evidence for an essential arginine in the flavoprotein Nitroalkane oxidase
Journal of enzyme inhibition, 2001Co-Authors: Giovanni Gadda, Ari Banerjee, Gaylon S. Fleming, Paul F. FitzpatrickAbstract:The flavoprotein Nitroalkane oxidase from the fungus Fusarium oxysporum catalyzes the oxidative denitrification of primary or secondary Nitroalkanes to yield the respective aldehydes or ketones, hydrogen peroxide and nitrite. The enzyme is inactivated in a time-dependent fashion upon treatment with the arginine-directed reagents phenylglyoxal, 2,3-butanedione, and cyclohexanedione. The inactivation shows first order kinetics with all reagents. Valerate, a competitive inhibitor of the enzyme, fully protects the enzyme from inactivation, indicating that modification is active site directed. The most rapid inactivation is seen with phenylglyoxal, with a kinact of 14.3 ± 1.1 M−1 min−1 in phosphate buffer at pH 7.3 and 30 °C. The lack of increase in the enzymatic activity of the phenylglyoxal-inactivated enzyme after removing the unreacted reagent by gel filtration is consistent with inactivation being due to co-valent modification of the enzyme. A possible role for an active site arginine in substrate binding...
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identification of a cysteine residue in the active site of Nitroalkane oxidase by modification with n ethylmaleimide
Journal of Biological Chemistry, 2000Co-Authors: Giovanni Gadda, Ari Banerjee, Lawrence J Dangott, Paul F. FitzpatrickAbstract:Abstract The flavoprotein Nitroalkane oxidase catalyzes the oxidative denitrification of primary or secondary Nitroalkanes to the corresponding aldehydes or ketones with production of hydrogen peroxide and nitrite. The enzyme is irreversibly inactivated by treatment with N-ethylmaleimide at pH 7. The inactivation is time-dependent and shows first-order kinetics for three half-lives. The second-order rate constant for inactivation is 3.4 ± 0.06 m − 1min− 1. The competitive inhibitor valerate protects the enzyme from inactivation, indicating an active site-directed modification. Comparison of tryptic maps of enzyme treated withN-[ethyl-1-14C]maleimide in the absence and presence of valerate shows a single radioactive peptide differentially labeled in the unprotected enzyme. The sequence of this peptide was determined to be LLNEVMCYPLFDGGNIGLR using Edman degradation and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The cysteine residue was identified as the site of alkylation by ion trap mass spectrometry.
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use of ph and kinetic isotope effects to dissect the effects of substrate size on binding and catalysis by Nitroalkane oxidase
Archives of Biochemistry and Biophysics, 2000Co-Authors: Giovanni Gadda, Damon Choe, Paul F. FitzpatrickAbstract:The flavoprotein Nitroalkane oxidase catalyzes the oxidation of a broad range of primary and secondary Nitroalkanes to the respective aldehydes or ketones, with production of hydrogen peroxide and nitrite. The V/K values for primary Nitroalkanes increase with increasing chain length, reaching a maximum with 1-nitrobutane [Gadda, G., and Fitzpatrick, P. F. (1999) Arch. Biochem. Biophys. 363, 309–313]. In the present report, pH and deuterium kinetic isotope effects with a series of primary Nitroalkanes and phenylnitromethane as substrates have been used to dissect the effects of chain length on binding and catalysis. The apparent pKa value for a group that must be unprotonated for catalysis decreases from about 7 to 5.3 with increasing size of the substrate. The D(V/K) values for these substrates decrease from 7.5 with nitroethane to 1 with phenylnitromethane. These results show that increasing the size of the substrate results in an increased partitioning forward to catalysis. The D(V/K) and DVmax values at pH 5.5 have been used to calculate the effect of substrate size on the Kd values for primary Nitroalkanes. The Kd values decrease with increasing length of the substrate, with a ΔΔGbinding of 1.7 kcal mol−1 for each additional methylene group. Such a value is less than the value of 2.6 kcal mol−1 previously determined for the effect of a methylene group on the V/K value [Gadda, G., and Fitzpatrick, P. F. (1999) Arch. Biochem. Biophys. 363, 309–313], suggesting that the total energy available per methylene group is used not only to enhance binding but also to increase the rate of catalysis.
Michael P. Valley - One of the best experts on this subject based on the ideXlab platform.
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characterization of active site residues of Nitroalkane oxidase
Bioorganic Chemistry, 2010Co-Authors: Michael P. Valley, Nana S Fenny, Shah R Ali, Paul F. FitzpatrickAbstract:The flavoenzyme Nitroalkane oxidase catalyzes the oxidation of primary and secondary Nitroalkanes to the corresponding aldehydes and ketones plus nitrite. The structure of the enzyme shows that Ser171 forms a hydrogen bond to the flavin N5, suggesting that it plays a role in catalysis. Cys397 and Tyr398 were previously identified by chemical modification as potential active site residues. To more directly probe the roles of these residues, the S171A, S171V, S171T, C397S, and Y398F enzymes have been characterized with nitroethane as substrate. The C397S and Y398 enzymes were less stable than the wild-type enzyme, and the C397S enzyme routinely contained a substoichiometric amount of FAD. Analysis of the steady-state kinetic parameters for the mutant enzymes, including deuterium isotope effects, establishes that all of the mutations result in decreases in the rate constants for removal of the substrate proton by approximately 5-fold and decreases in the rate constant for product release of approximately 2-fold. Only the S171V and S171T mutations alter the rate constant for flavin oxidation. These results establish that these residues are not involved in catalysis, but rather are required for maintaining the protein structure.
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Crystal structures of intermediates in the Nitroalkane oxidase reaction.
Biochemistry, 2009Co-Authors: Annie Heroux, Paul F. Fitzpatrick, Dragana M. Bozinovski, Michael P. Valley, Allen M. OrvilleAbstract:The flavoenzyme Nitroalkane oxidase is a member of the acyl-CoA dehydrogenase superfamily. Nitroalkane oxidase catalyzes the oxidation of neutral Nitroalkanes to nitrite and the corresponding aldehydes or ketones. Crystal structures to 2.2 A resolution or better of enzyme complexes with bound substrates and of a trapped substrate-flavin adduct are described. The D402N enzyme has no detectable activity with neutral Nitroalkanes [Valley, M. P., and Fitzpatrick, P. F. (2003) J. Am. Chem. Soc. 125, 8738-8739]. The structure of the D402N enzyme crystallized in the presence of 1-nitrohexane or 1-nitrooctane shows the presence of the substrate in the binding site. The aliphatic chain of the substrate extends into a tunnel leading to the enzyme surface. The oxygens of the substrate nitro group interact both with amino acid residues and with the 2'-hydroxyl of the FAD. When Nitroalkane oxidase oxidizes Nitroalkanes in the presence of cyanide, an electrophilic flavin imine intermediate can be trapped [Valley, M. P., Tichy, S. E., and Fitzpatrick, P. F. (2005) J. Am. Chem. Soc. 127, 2062-2066]. The structure of the enzyme trapped with cyanide during oxidation of 1-nitrohexane shows the presence of the modified flavin. A continuous hydrogen bond network connects the nitrogen of the CN-hexyl-FAD through the FAD 2'-hydroxyl to a chain of water molecules extending to the protein surface. Together, our complementary approaches provide strong evidence that the flavin cofactor is in the appropriate oxidation state and correlates well with the putative intermediate state observed within each of the crystal structures. Consequently, these results provide important structural descriptions of several steps along the Nitroalkane oxidase reaction cycle.
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mechanistic and structural analyses of the roles of arg409 and asp402 in the reaction of the flavoprotein Nitroalkane oxidase
Biochemistry, 2008Co-Authors: Paul F. Fitzpatrick, Annie Heroux, Dragana M. Bozinovski, Michael P. Valley, Patrick G Shaw, Allen M. OrvilleAbstract:The flavoprotein Nitroalkane oxidase (NAO) catalyzes the oxidation of primary and secondary Nitroalkanes to the corresponding aldehydes and ketones. The enzyme is a homologue of acyl-CoA dehydrogenase. Asp402 in NAO has been proposed to be the active site base responsible for removing the substrate proton in the first catalytic step; structurally it corresponds to the glutamate which acts as the base in medium chain acyl-CoA dehydrogenase. In the active site of NAO, the carboxylate of Asp402 forms an ionic interaction with the side chain of Arg409. The R409K enzyme has now been characterized kinetically and structurally. The mutation results in a decrease in the rate constant for proton abstraction of 100-fold. Analysis of the three-dimensional structure of the R409K enzyme, determined by X-ray crystallography to a resolution of 2.65 Angstroms, shows that the critical structural change is an increase in the distance between the carboxylate of Asp402 and the positively charged nitrogen in the side chain of the residue at position 409. The D402E mutation results in a smaller decrease in the rate constant for proton abstraction of 18-fold. The structure of the D402E enzyme, determined at 2.4 Angstroms resolution, shows that there is a smaller increase in the distancemore » between Arg409 and the carboxylate at position 402, and the interaction of this residue with Ser276 is perturbed. These results establish the critical importance of the interaction between Asp402 and Arg409 for proton abstraction by Nitroalkane oxidase.« less
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crystal structures of Nitroalkane oxidase insights into the reaction mechanism from a covalent complex of the flavoenzyme trapped during turnover
Biochemistry, 2006Co-Authors: A. K. Nagpal, Paul F. Fitzpatrick, Michael P. Valley, Allen M. OrvilleAbstract:Nitroalkane oxidase (NAO) from Fusarium oxysporum catalyzes the oxidation of neutral Nitroalkanes to the corresponding aldehydes or ketones with the production of H2O2 and nitrite. The flavoenzyme is a new member of the acyl-CoA dehydrogenase (ACAD) family, but it does not react with acyl-CoA substrates. We present the 2.2 A resolution crystal structure of NAO trapped during the turnover of nitroethane as a covalent N5−FAD adduct (ES*). The homotetrameric structure of ES* was solved by MAD phasing with 52 Se-Met sites in an orthorhombic space group. The electron density for the N5-(2-nitrobutyl)-1,5-dihydro-FAD covalent intermediate is clearly resolved. The structure of ES* was used to solve the crystal structure of oxidized NAO at 2.07 A resolution. The c axis for the trigonal space group of oxidized NAO is 485 A, and there are six subunits (11/2 holoenzymes) in the asymmetric unit. Four of the active sites contain spermine (EI), a weak competitive inhibitor, and two do not contain spermine (Eox). The ac...
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Establishing the kinetic competency of the cationic imine intermediate in Nitroalkane oxidase.
Journal of the American Chemical Society, 2005Co-Authors: Michael P. Valley, Shane E. Tichy, Paul F. FitzpatrickAbstract:The flavoprotein Nitroalkane oxidase catalyzes the oxidation of neutral Nitroalkanes to the corresponding aldehydes and ketones. Cyanide inactivates the enzyme during turnover in a concentration-dependent fashion. Mass spectrometry of the flavin from enzyme inactivated by cyanide in the presence of nitroethane or nitrohexane shows that a flavin cyanoethyl or cyanohexyl intermediate has formed. At high concentrations of cyanide, inactivation does not consume oxygen. Rapid reaction studies show that formation of the adduct with 2-2H2-nitroethane shows a kinetic isotope effect of 7.9. These results are consistent with cyanide reacting with a species formed after proton abstraction but before flavin oxidation. The proposed mechanism for Nitroalkane oxidase involves removal of a proton from the Nitroalkane, forming a carbanion which adds to the flavin N(5). Elimination of nitrite from the resulting adduct would form an electrophilic imine which can be attacked by hydroxide. The present results are consistent w...
Emerich S. Fiala - One of the best experts on this subject based on the ideXlab platform.
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Comparison of Oxidative Damage to Rat Liver DNA and RNA by Primary Nitroalkanes, Secondary Nitroalkanes, Cyclopentanone Oxime, and Related Compounds1
2013Co-Authors: C. Clifford Conaway, Guo Nie, S. Hussain, Cancer Res, Contact The Aacr Publications, Emerich S. FialaAbstract:The hepatocarcinogen 2-nitropropane causes oxidative damage to liver DNA and RNA after administration to rats; increases in 8-hydroxydeoxyguanosine and formation of an unknown moiety (DM) in DNA, plus increases in 8-hydroxyguanosine and the appearance of two unidentified peaks (KM and R\2) in RNA were observed by high-performance liquid chromatography of nucleosides from 2-nitropropane-treated rats using electrochemical detection (E. S. Fiala et al.. Cancer Res., 49:5518-5522, 1989). In the present study, damage to Sprague-Dawley rat liver RNA and DNA was assessed to determine whether the characteristic pattern of oxidative nucleic acid damage caused by 2-nitropropane also occurred after i.p. administration of primary Nitroalkanes, other secondary Nitroalkanes, 2-methyl-2-nitropropane (a tertiary Nitroalkane), and cyclopentanone oxime. All of the secondary Nitroalkanes and cyclopentanone oxime significantly increased levels of 8-hydroxyguanine in both DNA and RN
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Secondary Nitroalkanes: Induction of DNA repair in rat hepatocytes, activation by aryl sulfotransferase and hepatocarcinogenicity of 2-nitrobutane and 3-nitropentane in male F344 rats
Toxicology, 1995Co-Authors: Emerich S. Fiala, Rama S. Sodum, Nalband Hussain, Abraham Rivenson, Lisa DolanAbstract:The secondary Nitroalkanes, 2-nitropropane, 2-nitrobutane, 3-nitropentane, 2-nitroheptane, nitrocyclopentane and nitrocyclohexane, as well as the primary Nitroalkanes, 1-nitropropane, 1-nitrobutane, 1-nitropentane and 1-nitroheptane, were examined for their ability to induce DNA repair in rat hepatocytes and to serve as substrates for activation by partially purified rat liver aryl sulfotransferase in vitro. All of the secondary, but none of the primary Nitroalkanes examined, induced significant DNA repair in rat hepatocytes. Also, the nitronates of all of the secondary Nitroalkanes, but none of the primary Nitroalkanes, served as substrates for the aryl sulfotransferase-catalysed production of 8-aminoguanosine and 8-oxoguanosine from guanosine in vitro. In a carcinogenicity assay using male F344 rats, the secondary Nitroalkanes, 2-nitrobutane and 3-nitropentane, produced a highly significant incidence of hepatocarcinoma with metastases to the lungs, whereas the primary Nitroalkane, 1-nitrobutane, was not carcinogenic. While a low incidence of hepatocarcinoma was also produced by cyclopentanone oxime, the results were not statistically significant. Since the secondary Nitroalkane, 2-nitropropane, in contrast to the primary Nitroalkane, 1-nitropropane, was also previously shown to be hepatocarcinogenic in rats, it is probable that secondary Nitroalkanes constitute a hitherto unrecognized class of chemical carcinogens.
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Comparison of Oxidative Damage to Rat Liver DNA and RNA by Primary Nitroalkanes, Secondary Nitroalkanes, Cyclopentanone Oxime, and Related Compounds
Cancer research, 1991Co-Authors: C. Clifford Conaway, Nalband Hussain, Guo Nie, Emerich S. FialaAbstract:The hepatocarcinogen 2-nitropropane causes oxidative damage to liver DNA and RNA after administration to rats; increases in 8-hydroxydeoxyguanosine and formation of an unknown moiety (DX1) in DNA, plus increases in 8-hydroxyguanosine and the appearance of two unidentified peaks (RX1 and RX2) in RNA were observed by high-performance liquid chromatography of nucleosides from 2-nitropropane-treated rats using electrochemical detection (E. S. Fiala et al, Cancer Res., 49:5518-5522, 1989). In the present study, damage to Sprague-Dawley rat liver RNA and DNA was assessed to determine whether the characteristic pattern of oxidative nucleic acid damage caused by 2-nitropropane also occurred after i.p. administration of primary Nitroalkanes, other secondary Nitroalkanes, 2-methyl-2-nitropropane (a tertiary Nitroalkane), and cyclopentanone oxime. All of the secondary Nitroalkanes and cyclopentanone oxime significantly increased levels of 8-hydroxyguanine in both DNA and RNA and caused the appearance of DX1, RX1 and RX2. The primary Nitroalkanes and the tertiary Nitroalkane 2-methyl-2-nitropropane did not cause a similar pattern of nucleic acid damage. The rates of reprotonation of nitronates of the secondary Nitroalkanes to the respective un-ionized neutral forms at pH 7.7 were more than 20-fold less than the rates of reprotonation of primary Nitroalkane nitronates, suggesting that the anionic nitronates, rather than neutral compounds, are more immediately responsible for the DNA and RNA damage observed in vivo. Since 8-hydroxyguanine is a miscoding lesion in DNA, these results suggest the possibility, still to be rigorously tested, that hepatocarcinogenicity may be associated not only with 2-nitropropane but also with other secondary Nitroalkanes as well as with those ketoximes that are capable of being converted to secondary Nitroalkanes in vivo.
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Evaluation of secondary Nitroalkanes, their nitronates, primary Nitroalkanes, nitrocarbinols, and other aliphatic nitro compounds in the Ames Salmonella assay.
Mutation research, 1991Co-Authors: C. Clifford Conaway, Nalband Hussain, Barbara M. Way, Emerich S. FialaAbstract:Abstract The secondary Nitroalkanes 2-nitropropane, 2-nitrobutane, 3-nitropentane and nitrocyclopentane, as well as their anionic forms (nitronates); the primary Nitroalkanes 1-nitropropane, 1-nitrobutane, and 1-nitropentane and their respective nitronates; the nitrocarbinols 2-nitro-1-propanol, 2-nitro-1-butanol, 3-nitro-2-butanol, and 3-nitro-2-pentanol and their respective nitronates; 2-methyl-2-nitropropane, and 2-nitroso-2-nitropropane were tested in the Ames Salmonella assay using strains TA98, TA100 and TA102. Nitronates of the secondary Nitroalkanes 2-nitropropane, 2-nitrobutane, 3-nitropentane, and nitrocyclopentane were significantly mutagenic in Salmonella strains TA100 and TA102 at 10–80 μ moles/plate, but the parent compounds were mutagenic at only a single dose level or were not mutagenic at all in the same dose range. The primary Nitroalkanes and the nitrocarbinols were not mutagenic, or only marginally so, at the concentrations tested. The nitronates of the primary Nitroalkanes and the nitrocarbinols reprotonated too rapidly under the conditions of the assay for adequate evaluation of mutagenicity. 2-Methyl-2-nitropropane was not mutagenic in strains TA100 and TA102; 2-nitroso-2-nitropropane was also not mutagenic in strains TA100 and TA102, but induced an equivocal mutagenic response in TA98. The positive Salmonella mutation data for the nitronates of the secondary Nitroalkanes studied correlate very well with the very slow rate of reprotonation of secondary Nitroalkane nitronates at pH 7.7 (Conaway et al. (1991) Cancer Res., 51, 3143), and provide further evidence that nitronates of secondary Nitroalkanes, rather than the neutral parent forms with which they may be in equilibrium, are the more proximate mutagenic species.
Huarong Tan - One of the best experts on this subject based on the ideXlab platform.
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Crystal structure and site-directed mutagenesis of a Nitroalkane oxidase from Streptomyces ansochromogenes
Biochemical and biophysical research communications, 2010Co-Authors: Zengqiang Gao, Haifeng Hou, Jihui Zhang, Haihua Yang, Yuhui Dong, Huarong TanAbstract:Nitroalkane oxidase (NAO) catalyzes neutral Nitroalkanes to their corresponding aldehydes or ketones, hydrogen peroxide and nitrite. The crystal structure of NAO from Streptomyces ansochromogenes was determined; it consists of two domains, a TIM barrel domain bound to FMN and C-terminal domain with a novel folding pattern. Site-directed mutagenesis of His179, which is spatially adjacent to FMN, resulted in the loss of enzyme activity, demonstrating that this amino acid residue is important for catalysis. The crystal structure of mutant H179D-nitroethane was also analyzed. Interestingly, Sa-NAO shows the typical function as Nitroalkane oxidase but its structure is similar to that of 2-nitropropane dioxygenase. Overall, these results suggest that Sa-NAO is a novel Nitroalkane oxidase with TIM barrel structure.
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identification of a Nitroalkane oxidase gene naoa related to the growth of streptomyces ansochromogenes
Current Microbiology, 2008Co-Authors: Jihui Zhang, Huarong TanAbstract:naoA, encoding a Nitroalkane oxidase that can catalyze toxic Nitroalkanes to their corresponding aldehydes or ketones and hydrogen peroxide, was cloned from Streptomyces ansochromogenes, but its function related to the growth of Streptomyces is unknown. naoA was disrupted by the insertion of a kanamycin-resistance gene; the resulting strain can grow earlier than a wild-type strain under the same conditions. It was shown that naoA disruption accelerated growth of the naoA-disruption mutant, which could restore its phenotype and morphology as a wild-type strain by complementation of a single copy number of naoA inserted into the chromosome. The introduction of an extra copy of naoA into the wild-type strain resulted in delayed growth. The result suggested that naoA is an important gene related to the growth of S. ansochromogenes.