Reaction of Phthalate Dioxygenase Reductase with NADH and NAD: Kinetic and Spectral Characterization of Intermediates

Gassner, George; Wang, Lihua; Batie, Christopher J.; Ballou, David P.

doi:10.1021/bi00206a022

Cited by 54 publications

(67 citation statements)

References 45 publications

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“…It is highly unlikely that NG could form a similar adduct as part of the denitration reaction, particularly in light of the demonstration that NG reacts with the reduced flavin. In contrast, the zwitterionic nature of the electrondeficient nitroester functional group could allow formation of a charge transfer intermediate with an electron-rich reduced flavin, such as has been recently discussed for flavoprotein NADH oxidoreductases (17). This charge transfer complex could then permit electron transfers and the subsequent cleavage of the nitroester.…”

Section: Discussionmentioning

confidence: 99%

Regioselectivity of nitroglycerin denitration by flavoprotein nitroester reductases purified from two Pseudomonas species

et al. 1997

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Two species of Pseudomonas capable of utilizing nitroglycerin (NG) as a sole nitrogen source were isolated from NG-contaminated soil and identified as Pseudomonas putida II-B and P. fluorescens I-C. While 9 of 13 laboratory bacterial strains that presumably had no previous exposure to NG could degrade low concentrations of NG (0.44 mM), the natural isolates tolerated concentrations of NG that were toxic to the lab strains (1.76 mM and higher). Whole-cell studies revealed that the two natural isolates produced different mixtures of the isomers of dinitroglycerol (DNG) and mononitroglycerol (MNG). A monomeric, flavin mononucleotide-containing NG reductase was purified from each natural isolate. These enzymes catalyzed the NADPH-dependent denitration of NG, yielding nitrite. Apparent kinetic constants were determined for both reductases. The P. putida enzyme had a K m for NG of 52 ؎ 4 M, a K m for NADPH of 28 ؎ 2 M, and a V max of 124 ؎ 6 M ⅐ min ؊1 , while the P. fluorescens enzyme had a K m for NG of 110 ؎ 10 M, a K m for NADPH of 5 ؎ 1 M, and a V max of 110 ؎ 11 M ⅐ min ؊1 . Anaerobic titration experiments confirmed the stoichiometry of NADPH consumption, changes in flavin oxidation state, and multiple steps of nitrite removal from NG. The products formed during time-dependent denitration reactions were consistent with a single enzyme being responsible for the in vivo product distributions. Simulation of the product formation kinetics by numerical integration showed that the P. putida enzyme produced an Ϸ2-fold molar excess of 1,2-DNG relative to 1,3-DNG. This result could be fortuitous or could possibly be consistent with a random removal of the first nitro group from either the terminal (C-1 and C-3) positions or middle (C-2) position. However, during the denitration of 1,2-DNG, a 1.3-fold selectivity for the C-1 nitro group was determined. Comparable simulations of the product distributions from the P. fluorescens enzyme showed that NG was denitrated with a 4.6-fold selectivity for the C-2 position. Furthermore, a 2.4-fold selectivity for removal of the nitro group from the C-2 position of 1,2-DNG was also determined. The MNG isomers were not effectively denitrated by either purified enzyme, which suggests a reason why NG could not be used as a sole carbon source by the isolated organisms.Nitroglycerin (NG, glycerol trinitrate) is an aliphatic nitrate ester-containing compound that is an important component of dynamite and other propellants (36,42). In addition, nitrosubstituted compounds, including nitroaromatics, are used as explosives, agricultural chemicals, and dyes (18, 32, 39). Consequently, compounds containing nitro functional groups have become widely distributed in the environment. Since naturally occurring nitro functional groups are rare (18, 32), the molecules containing these groups have typically been classified as xenobiotics. Consistent with this classification, a number of early studies of the environmental fate of NG revealed toxicity to algae, invertebrates, and vertebrates (42) and furth...

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Section: Discussionmentioning

confidence: 99%

Regioselectivity of nitroglycerin denitration by flavoprotein nitroester reductases purified from two Pseudomonas species

et al. 1997

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“…Although there is no direct evidence for the conversion of E-FAD-NADH to E-FAD-NADH* (process (ii)), the existence of the E-FAD-NADH complex, which has no ability to transfer H Ϫ , is a reasonable assumption. This is because H Ϫ transfer itself is generally very fast, and a two-step mechanism for pyridine nucleotide binding has been proposed for the related family enzymes, nitrate reductase (50), phthalate dioxygenase reductase (51,52), and FNR (31). In the WT and the T66S mutant, the rate-limiting step is process (ii).…”

Section: Discussionmentioning

confidence: 99%

Role of Thr66 in Porcine NADH-cytochromeb 5 Reductase in Catalysis and Control of the Rate-limiting Step in Electron Transfer

Kimura

Kawamura

Iyanagi

2003

Journal of Biological Chemistry

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Site-directed mutagenesis of Thr 66 in porcine liver NADH-cytochrome b 5 reductase demonstrated that this residue modulates the semiquinone form of FAD and the rate-limiting step in the catalytic sequence of electron transfer. The absorption spectrum of the T66V mutant showed a typical neutral blue semiquinone intermediate during turnover in the electron transfer from NADH to ferricyanide but showed an anionic red semiquinone form during anaerobic photoreduction. The apparent k cat values of this mutant were ϳ10% of that of the wild type enzyme (WT). These data suggest that the T66V mutation stabilizes the neutral blue semiquinone and that the conversion of the neutral blue to the anionic red semiquinone form is the rate-limiting step. In the WT, the value of the rate constant of FAD reduction (k red ) was consistent with the k cat values, and the oxidized enzyme-NADH complex was observed during the turnover with ferricyanide. This indicates that the reduction of FAD by NADH in the WT-NADH complex is the ratelimiting step. In the T66A mutant, the k red value was larger than the k cat values, but the k red value in the presence of NAD ؉ was consistent with the k cat values. The spectral shape of this mutant observed during turnover was similar to that during the reduction with NADH in the presence of NAD ؉ . These data suggest that the oxidized T66A-NADH-NAD ؉ ternary complex is a major intermediate in the turnover and that the release of NAD ؉ from this complex is the rate-limiting step. These results substantiate the important role of Thr 66 in the one-electron transfer reaction catalyzed by this enzyme. On the basis of these data, we present a new kinetic scheme to explain the mechanism of electron transfer from NADH to one-electron acceptors including cytochrome b 5 .NADH-cytochrome b 5 reductase (EC 1.6.2.2) is a member of the large family of flavin-dependent oxidoreductases that transfer an electron from two-electron carriers of nicotinamide dinucleotides to one-electron carriers such as heme proteins and ferredoxins. This enzyme catalyzes the electron transfer from NADH to cytochrome b 5 (b5) 1 (1-3), and participates in fatty acid synthesis (4, 5), cholesterol synthesis (6), and xenobiotic oxidation (7) as a member of the electron transport chain on the endoplasmic reticulum. In erythrocytes, this enzyme participates in the reduction of methemoglobin (8).The outline of the catalytic cycle of the solubilized catalytic domain of NADH-cytochrome b 5 reductase (b5R) is understood as follows (3) (Scheme I). At first, two electrons are transferred from NADH to FAD by hydride (H Ϫ ) transfer. Then the twoelectron reduced enzyme-NAD ϩ complex (E-FADH Ϫ -NAD ϩ ) transfers two electrons to two one-electron acceptors one by one via the anionic red semiquinone form (E-FAD⅐ Ϫ -NAD ϩ ), and the reduced enzyme returns to the oxidized state. Strittmatter (9 -11) suggested that the reduction of FAD by NADH is the rate-limiting step in electron transfer catalyzed by b5R. Iyanagi et al. (3,12) found that the anionic red sem...

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“…A conformational change was postulated in order to allow direct contact of the nicotinamide ring and the flavin (38). This is supported by rapid kinetic studies on PDR, which suggested a two-step mechanism for NADH binding (25,26).…”

Section: Atp-ribosementioning

confidence: 94%

The NAD(P)H:Flavin Oxidoreductase from Escherichia coli

Nivière

Fieschi

Décout

et al. 1999

Journal of Biological Chemistry

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The NAD(P)H:flavin oxidoreductase from Escherichia coli, named Fre, is a monomer of 26.2 kDa that catalyzes the reduction of free flavins using NADPH or NADH as electron donor. The enzyme does not contain any prosthetic group but accommodates both the reduced pyridine nucleotide and the flavin in a ternary complex prior to oxidoreduction. The specificity of the flavin reductase for the pyridine nucleotide was studied by steady-state kinetics using a variety of NADP analogs. Both the nicotinamide ring and the adenosine part of the substrate molecule have been found to be important for binding to the polypeptide chain. However, in the case of NADPH, the 2-phosphate group destabilized almost completely the interaction with the adenosine moiety. Moreover, NADPH and NMNH are very good substrates for the flavin reductase, and we have shown that both these molecules bind to the enzyme almost exclusively by the nicotinamide ring. This provides evidence that the flavin reductase exhibits a unique mode for recognition of the reduced pyridine nucleotide. In addition, we have shown that the flavin reductase selectively transfers the pro-R hydrogen from the C-4 position of the nicotinamide ring and is therefore classified as an A-side-specific enzyme.Flavin reductases are enzymes defined by their ability to catalyze the reduction of free flavins (riboflavin, FMN, or FAD) by using reduced pyridine nucleotides, NADPH, or NADH (1). The products of these enzyme activities, protein-free reduced flavins, were suggested to have important biological functions as electron transfer mediators, even though the real physiological significance of these mediators has so far not been fully appreciated. In fact, in vitro, free reduced flavins can reduce ferric complexes or iron proteins very efficiently, and it has been suggested that flavin reductases could play a key role in: iron metabolism (1, 2), activation of ribonucleotide reductase (3, 4), and reduction of methemoglobin (5, 6). There is also indirect evidence for their function in bioluminescence (7,8) and oxygen reduction (9). Recently, flavin reductases have been found to be associated with oxygenases involved in the desulfurization process of fossil fuels (10) and antibiotic biosynthesis (11-13).Up to now, two major classes of flavin reductases have been characterized. Class I enzymes do not contain any flavin prosthetic group and cannot be defined as flavoproteins (3,14), whereas class II enzymes are canonical flavoproteins (15-17). These two classes also exhibit different enzymatic mechanisms for flavin reduction. Class I enzymes use a sequential mechanism (14), whereas class II use a Ping-Pong mechanism, as a consequence of having a redox cofactor bound to the polypeptide chain (17, 18).The prototype for class I flavin reductases is an enzyme, named Fre, 1 which was initially discovered in Escherichia coli as a component of a multienzymatic system involved in the activation of ribonucleotide reductase, a key enzyme in DNA biosynthesis (3, 4). Fre consists of a single polypept...

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Reaction of Phthalate Dioxygenase Reductase with NADH and NAD: Kinetic and Spectral Characterization of Intermediates

Cited by 54 publications

References 45 publications

Regioselectivity of nitroglycerin denitration by flavoprotein nitroester reductases purified from two Pseudomonas species

Regioselectivity of nitroglycerin denitration by flavoprotein nitroester reductases purified from two Pseudomonas species

Role of Thr66 in Porcine NADH-cytochromeb 5 Reductase in Catalysis and Control of the Rate-limiting Step in Electron Transfer

The NAD(P)H:Flavin Oxidoreductase from Escherichia coli

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