Purification and properties of NADH-ferredoxinTOL reductase. A component of toluene dioxygenase from Pseudomonas putida.

Subramanian, V.; Liu, T N; Yeh, W K; Narro, Martha L.; Gibson, David T.

doi:10.1016/s0021-9258(19)69675-4

Cited by 94 publications

(4 citation statements)

References 31 publications

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“…The flavin content was also quantitated spectrophotometrically (e456 = 1 1.3 mM-1 cm-1) after heat denaturation of the reductase. Metal chelators and sulfhydryl alkylating reagents were assayed for the ability to inhibit reductase activity (Subramanian et al, 1981). Isoelectric point determinations were performed using a Phast-gel System and IEF 3-9 media (Pharmacia).…”

Section: Methodsmentioning

confidence: 99%

Purification and characterization of toluene 2-monooxygenase from Burkholderia cepacia G4

1995

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Recent in vivo studies indicate that ring monooxygenation is a widespread mechanism by which bacteria metabolize aromatic hydrocarbons and obtain carbon and energy. In this study, toluene 2-monooxygenase from Burkholderia (formerly Pseudomonas) cepacia G4 was purified to homogeneity and found to be a three-component enzyme system. The reconstituted enzyme system oxidized toluene to o-cresol and o-cresol to 3-methylcatechol, an important intermediate for growth of the bacterium on toluene. Steady-state kinetic parameters measured for the water-soluble substrate o-cresol were a Km of 0.8 microM and a Vmax of 131 nmol min-1 (mg of hydroxylase protein)-1. The three protein components were (1) a 40 kDa polypeptide containing one FAD and a [2Fe2S] cluster, (2) a 10.4 kDa polypeptide that contained no identifiable metals or organic cofactors, and (3) a 211 kDa alpha 2 beta 2 gamma 2 component containing five to six iron atoms. The 40 kDa flavo-iron-sulfur protein oxidized NADH and transferred electrons to cytochrome c, dyes, and the alpha 2 beta 2 gamma 2 component. It is analogous to other NADH oxidoreductase components found in a wide range of bacterial mono- and dioxygenases. The 10.4 kDa component, added to the other two components and NADH, increased toluene oxidation rates 10-fold. The alpha 2 beta 2 gamma 2 component was indicated to contain the site for toluene binding and hydroxylation by the following observations: (1) tight binding to a toluene affinity column; (2) oxidation of toluene after reduction of the protein with dithionite and adding O2; (3) H2O2-dependent toluene oxidation and catalase activity; and (4) spectroscopic studies of the iron atoms in the component. The alpha 2 beta 2 gamma 2 component had no significant absorbance in the visible region. EPR spectroscopy yielded a signal at g = 16 upon addition of > 2 equiv of electrons per 2 Fe atoms. Taken with the quantitation of five to six iron atoms, the data suggest that the alpha 2 beta 2 gamma 2 component contains two binuclear iron centers. In total, the structural, spectroscopic, and catalytic features of toluene 2-monooxygenase are reminiscent of soluble methane monooxygenase obtained from methanotrophic bacteria. The two enzyme systems also differ in many subtle ways; for example, they oxidize toluene with completely different regiospecificity.

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Section: Methodsmentioning

confidence: 99%

Purification and characterization of toluene 2-monooxygenase from Burkholderia cepacia G4

1995

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“…Toluene Dioxygenase and Naphthalene Dioxygenase. The three components of the toluene dioxygenase enzyme system were purified as previously described (Subramanian et al, 1979(Subramanian et al, , 1981. These were ferredoxinXOL reductase, ferredox-inTOL, and the terminal oxygenase (ISPXOl)• The first two enzymes gave single bands that stained for protein when analyzed on 12% polyacrylamide gels in the presence of sodium dodecyl sulfate.…”

Section: Methodsmentioning

confidence: 99%

“…Electrons are transferred from NADH1 through a flavoprotein (reductase-rot) and a [2Fe-2S] protein (ferredoxinTOL) to a terminal iron-sulfur protein (ISPX0L) that catalyzes the oxidation of toluene to cw-toluene dihydrodiol. Although these individual components have been characterized in some detail (Subramanian et al, 1981(Subramanian et al, , 1985Gibson et al, 1982), little is known about the oxygen insertion reaction.…”

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confidence: 99%

Benzylic monooxygenation catalyzed by toluene dioxygenase from Pseudomonas putida

Wackett¹,

Kwart²,

Gibson³

1988

Biochemistry

207

141

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Toluene dioxygenase, a multicomponent enzyme system known to oxidize mononuclear aromatic hydrocarbons to cis-dihydrodiols, oxidized indene and indan to 1-indenol and 1-indanol, respectively. In addition, the enzyme catalyzed dioxygen addition to the nonaromatic double bond of indene to form cis-1,2-indandiol. The oxygen atoms in 1-indenol and cis-1,2-indandiol were shown to be derived from molecular oxygen, whereas 70% of the oxygen in 1-indanol was derived from water. All of the isolated products were optically active as demonstrated by 19F NMR and HPLC discrimination of diastereomeric esters and by chiroptic methods. The high optical purity of (-)-(1R)-indanol (84% enantiomeric excess) and the failure of scavengers of reactive oxygen species to inhibit the monooxygenation reaction supported the contention that the monooxygen insertion is mediated by an active-site process. Experiments with 3-[2H]indene indicated that equilibration between C-1 and C-3 occurred prior to the formation of the carbon-oxygen bond to yield 1-indenol. Naphthalene dioxygenase also oxidized indan to 1-indanol, which suggested that benzylic monoxygenation may be typical of this group of dioxygenases.

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“…<6> Clostridium thermohydrosulfuricum (activity is present in wild-type strain, alcohol-adapted strain lacks detectable levels of reduced ferredoxin-linked NAD reductase [4]) [4] <7> Pseudomonas putida (NAD-ferredocinTOL reductase component of toluene dioxygenase [5]) [5] <8> Clostridium tyrobutyricum (strain CNRZ 510 [6]) [6,9] <9> Clostridium thermocellum [7] <10> Clostridium brockii [7] <11> Clostridium acetobutylicum [8,9] <12> Butyribacterium methylotrophicum [13] 3 Reaction and Specificity [8,9]; <8>, in cells growing on pyruvate/acetate NADH does not control enzyme activity [9]) [8,9] NEM <1> (<1>, 10 mM, 67% inhibition [1]) [1] PCMB <1, 7> (<1>, 0.0005 mM, 94% inhibition [1]) [1,5] iodoacetate <1> (<1>, 10 mM, 50% inhibition [1]) [1] sodium azide <1> (<1>, 40 mM, 46% inhibition [1]) [1] Cofactors/prosthetic groups FAD <1, 7> (<1>, 1 mol of FAD is bound per mol of enzyme [1]; <7>, can bind one mol of FAD per mol of enzyme, K m : 2.5 nM [5]; <1>, contains 0.89 mol of FAD per mol of enzyme [12]) [1,5,12] NADH <1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12> (<5>, specific for NADH [3]; <12>, the CO-adapted strain is a metabolic mutant having higher levels of ferredoxin-NAD oxidoreductase activity, which is not inhibited by NADH [13]) [1,2,3,…”

mentioning

confidence: 99%

Ferredoxin-NAD+ reductase

Springer Handbook of Enzymes

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Systematic name ferredoxin:NAD + oxidoreductase Recommended name ferredoxin-NAD + reductase Synonyms NAD-ferredocinTOL reductase <7> (<7>, component of toluene dioxygenase [5]) [5] NAD-ferredoxin reductase NADH flavodoxin oxidoreductase NADH-ferredoxin oxidoreductase NADH-ferredoxin reductase NADH-ferredoxinNAP reductase <1> (<1>, component of naphthalene dioxygenase multicomponent enzyme system [1]) [1] NADH 2 -ferredoxin oxidoreductase ferredoxin-NAD reductase ferredoxin-linked NAD reductase reductase, ferredoxin reductase, ferredoxin-nicotinamide adenine dinucleotide reductase, reduced nicotinamide adenine dinucleotide-ferredoxin CAS registry number 39369-37-4 2 Source Organism <1> Pseudomonas sp. (strain NCIB 9816 [1]; strain KKS102 [11]; strain LB400 [12]; NADH-ferredoxinNAP reductase component of naphthalene dioxygenase [1]; NADH-ferredoxin oxidoreductase component of biphenyl 2,3dioxygenase [12]) [1, 11, 12] <2> Clostridium kluyveri [2] <3> Clostridium pasteurianum [2, 6, 9, 10] <4> Clostridium butyricum [2, 10] <5> Methylosinus trichosporium OB3b [3]

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Purification and properties of NADH-ferredoxinTOL reductase. A component of toluene dioxygenase from Pseudomonas putida.

Cited by 94 publications

References 31 publications

Purification and characterization of toluene 2-monooxygenase from Burkholderia cepacia G4

Purification and characterization of toluene 2-monooxygenase from Burkholderia cepacia G4

Benzylic monooxygenation catalyzed by toluene dioxygenase from Pseudomonas putida

Ferredoxin-NAD+ reductase

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