Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: KEGG:D02011 (FAD)
 5,530 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Nitroalkane oxidase from Fusarium oxysporum catalyzes the oxidation of nitroalkanes to aldehydes with production of nitrite and hydrogen peroxide. The UV-visible absorbance spectrum of the purified enzyme shows a single absorption peak at 336 nm with an extinction coefficient of 7.4 mM-1 cm-1. Upon denaturation of the enzyme at pH 7.0, a stoichiometric amount of FAD is released. The spectral properties of the enzyme as isolated are consistent with an N(5) adduct of the flavin. This is not due to a covalent linkage with the protein, since the free flavin adduct can be isolated from the enzyme at pH 2.1. The free flavin adduct shows an absorbance spectrum with a lambdamax at 346 nm (10.7 mM-1 cm-1) and is not fluorescent. Under alkaline conditions the free adduct decays, yielding FAD; the rate of this process is pH-dependent with a pKa of 7.4. Adduct decay is also observed with the native enzyme; in this case, however, the rate of decay is 160-fold slower (at pH 8.0) and not dependent on pH. During this process a large increase in enzymatic activity ( approximately 26-fold at pH 7.0) is observed, the rate of which is equal to the rate of flavin adduct conversion to FAD. Thus, the native flavin adduct is not active but can be converted to FAD, the active form of the flavin. Maximal activation is pH- and FAD-dependent; two groups with pKa values of 5.65 +/- 0. 25 and 8.75 +/- 0.05 must be unprotonated and protonated, respectively. The m/z- of the free flavin adduct is 103.0645 higher than that of FAD, as determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. This corresponds to a molecule of nitrobutane linked to FAD. A mechanism is proposed for the formation in vivo of the nitrobutyl-FAD of nitroalkane oxidase.
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PMID:Identification of the naturally occurring flavin of nitroalkane oxidase from fusarium oxysporum as a 5-nitrobutyl-FAD and conversion of the enzyme to the active FAD-containing form. 903 63

Nitroalkane oxidase from Fusarium oxysporum catalyzes the oxidation of nitroalkanes to aldehydes with production of nitrite and hydrogen peroxide. The enzyme has a molecular weight of 47 955 +/- 39, as determined by MALDI-TOF mass spectrometry; under nondenaturing conditions, the aggregation state of the enzyme is best described by a tetramer-dimer self-associating model, with an association constant of (8.5 +/- 4.4) x 10(6) M-1 (pH 7.0 and 4 degreesC). The amino acid composition and the N-terminal amino acid sequence do not match any known protein or open reading frame. The inactive 5-nitrobutyl-1,5-dihydroflavin found in the enzyme as purified was converted to FAD, allowing characterization of the active FAD-containing enzyme. With nitroethane as substrate, the Vmax and Km values are 655 +/- 45 min-1 and 2.9 +/- 0.5 mM at pH 8.0 and 30 degreesC, respectively. One mole of FAD per mole of monomer enzyme is required for catalysis. No activity can be detected with amino acids or alpha-hydroxy acids as substrates. Reversible removal of the FAD cofactor yields inactive enzyme. The properties of the FAD cofactor in nitroalkane oxidase are within the range described for other oxidases. The UV-visible absorbance spectrum of the active enzyme shows maxima at 446, 384, and 274 nm; the extinction coefficient at 446 nm is 11.7 mM-1 cm-1. The neutral form of the flavin semiquinone, with maxima at 536 and 342 nm, is kinetically stabilized. The UV-visible absorbance spectrum of the reduced enzyme is typical of the anionic form of a flavin, with a peak centered at 335 nm. The affinity of the enzyme for sulfite is low (Kd value of 13.8 +/- 0.9 mM at pH 7.0 and 25 degreesC); this result, along with the stabilization of the neutral flavin semiquinone, suggests the presence of a weak positive charge near the N(1)-C(2)=O of FAD. The reduction potential of the enzyme is -367 mV. Benzoate and phenylacetic acid are competitive inhibitors, with Kis values of 5.1 +/- 0.6 and 13.1 +/- 2.3 mM, respectively. Binding of benzoate to nitroalkane oxidase results in spectral changes similar to those observed with d-amino acid oxidase. The absorbance spectrum of the flavin bound to nitroalkane oxidase is pH-dependent, with a pKa value of 8.4.
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PMID:Biochemical and physical characterization of the active FAD-containing form of nitroalkane oxidase from Fusarium oxysporum. 955 55

The flavoprotein nitroalkane oxidase from Fusarium oxysporum catalyzes the oxidation of nitroalkanes to aldehydes with production of hydrogen peroxide and nitrite. The substrate specificity of the FAD-containing enzyme has been determined as a probe of the active site structure. Nitroalkane oxidase is active on primary and secondary nitroalkanes, with a marked preference for unbranched primary nitroalkanes. The V/K values for primary nitroalkanes increase with increasing length of the alkyl chain, reaching a maximum with 1-nitrobutane, suggesting a hydrophobic binding site sufficient to accommodate a four carbon chain. Each methylene group of the substrate contributes approximately 2.6 kcal mol-1 in binding energy. The V/K values for substrates containing a hydroxyl group are two orders of magnitude smaller than those of the corresponding nitroalkanes, also consistent with a hydrophobic binding site. 3-Nitro-1-propionate is a competitive inhibitor with a Kis value of 3.1 +/- 0.2 mM.
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PMID:Substrate specificity of a nitroalkane-oxidizing enzyme. 1006 53

2-oxo-3-pentynoate has been characterized as an active-site-directed inhibitor of selected flavoprotein oxidases. Tryptophan 2-monooxygenase is irreversibly inactivated in an active-site-directed fashion. The addition of FAD affords no protection from inactivation, whereas the competitive inhibitor indole-3-acetamide fully protects the enzyme from inactivation. The inactivation follows first-order kinetics for at least five half-lives. The rate of inactivation shows saturation kinetics, consistent with the formation of a reversible complex between the alkylating agent and the enzyme before inactivation occurs. Values of 0.017 +/- 0.0005 min-1 and 44 +/- 7 microM were determined for the limiting rate of inactivation and the apparent dissociation constant for 2-oxo-3-pentynoate, respectively. Tryptic maps of tryptophan 2-monooxygenase treated with 2-oxo-3-pentynoate show that two peptides are alkylated in the absence of indole-3-acetamide but not in its presence. The two peptides were identified by mass spectrometry as residues 333-349 and 503-536. Based upon sequence analysis, cysteine 511 and either cysteine 339 or histidine 338 are the likely sites of modification. In contrast, incubation of D-amino acid oxidase or nitroalkane oxidase with 2-oxo-3-pentynoate results in a loss of 55% or 100%, respectively, of the initial activity. In neither case does a competitive inhibitor affect the rate of inactivation, suggesting that the effect is not due to modification of active-site residues.
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PMID:Characterization of 2-oxo-3-pentynoate as an active-site-directed inactivator of flavoprotein oxidases: identification of active-site peptides in tryptophan 2-monooxygenase. 1023 33

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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PMID:Cloning of nitroalkane oxidase from Fusarium oxysporum identifies a new member of the acyl-CoA dehydrogenase superfamily. 1186 31

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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PMID:Crystal structures of intermediates in the nitroalkane oxidase reaction. 1926 37

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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PMID:Characterization of active site residues of nitroalkane oxidase. 2005 14