Purification and properties of NADH-ferredoxinNAP reductase, a component of naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816

Haigler, B E; Gibson, D T

doi:10.1128/jb.172.1.457-464.1990

Cited by 152 publications

(118 citation statements)

References 43 publications

(25 reference statements)

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“…In contrast, it appears that neither oxidized nor reduced NDR increases the yield of NDO turnover, indicating that NDR is incapable of transferring electrons to NDO or transferring electrons between oxygenase active sites in the absence of NDF. This observation is consistent with the proposed role of NDF in coupling electron transfer from NADH-reduced NDR to NDO (39,40). Neither reduced NDF nor reduced NDR alone catalyze added.…”

Section: Resultssupporting

confidence: 80%

Single Turnover Chemistry and Regulation of O2Activation by the Oxygenase Component of Naphthalene 1,2-Dioxygenase

Wolfe¹,

Parales²,

Gibson³

et al. 2001

Journal of Biological Chemistry

173

311

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Naphthalene 1,2-dioxygenase (NDOS) is a three-component enzyme that catalyzes cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene formation from naphthalene, O 2 , and NADH. We have determined the conditions for a single turnover of NDOS for the first time and studied the regulation of catalysis. As isolated, the ␣ 3 ␤ 3 oxygenase component (NDO) has up to three catalytic pairs of metal centers (one mononuclear Fe 2؉ and one diferric Rieske iron-sulfur cluster). This form of NDO is unreactive with O 2 . However, upon reduction of the Rieske cluster and exposure to naphthalene and O 2 , ϳ0.85 cisdiol product per occupied mononuclear iron site rapidly forms. Substrate binding is required for oxygen reactivity. Stopped-flow and chemical quench analyses indicate that the rate constant of the single turnover product-forming reaction significantly exceeds the NDOS turnover number. UV-visible and electron paramagnetic resonance spectroscopies show that during catalysis, one mononuclear iron and one Rieske cluster are oxidized per product formed, satisfying the two-electron reaction stoichiometry. The addition of oxidized or reduced NDOS ferredoxin component (NDF) increases both the product yield and rate of oxidation of formerly unreactive Rieske clusters. The results show that NDO alone catalyzes dioxygenase chemistry, whereas NDF appears to serve only an electron transport role, in this case redistributing electrons to competent active sites.Rieske nonheme iron dioxygenases catalyze a remarkable reaction in which dioxygen is cleaved and both atoms are inserted across a double bond of an unactivated aromatic nucleus to yield a cis-dihydrodiol (1-4). These enzymes initiate the biodegradation of some of the most recalcitrant aromatic compounds that enter the environment from both natural and industrial sources, making their study of great importance for progress in bioremediation practices (5, 6). In addition, the inherent enantio-and regiospecificity of these enzymes make them useful for synthetic applications (7,8). Several of these multicomponent dioxygenases are known that differ in the number of components and the number and type of subunits in the oxygenase component that contains the active site. However, all of the enzyme systems share the common features of a reductase component that can accept and pass on two electrons from reduced pyridine nucleotide, an electron transport system that may be encompassed into the reductase, and an oxygenase that has both Rieske [2Fe-2S] clusters and mononuclear iron centers.One of the most thoroughly studied of the Rieske nonheme iron dioxygenases is naphthalene 1,2-dioxygenase (NDOS) 1 isolated from Pseudomonas sp. NCIB 9816-4, which catalyzes the reaction shown in Scheme 1 to yield (ϩ)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene (9).The complete NDOS consists of three protein components: the 36-kDa reductase protein (NDR) that contains a FAD molecule and a plant-type [2Fe-2S] cluster, the 14-kDa ferredoxin electron transfer protein (NDF) that contains a [2Fe-2S] Rieske cluster, an...

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Section: Resultssupporting

confidence: 80%

Single Turnover Chemistry and Regulation of O2Activation by the Oxygenase Component of Naphthalene 1,2-Dioxygenase

Wolfe¹,

Parales²,

Gibson³

et al. 2001

Journal of Biological Chemistry

173

311

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“…This corresponds to preliminary data from a partial purification of the desulfonative enzyme complex for BS in another bacterium (Kondo et al 1982). The idea is analogous to the suggestion of Brilon et al (1981a), that the desulfonation of naphthalene sulfonate is carried out by a mutant naphthalene dioxygenase with a wide substrate specificity; naphthalene dioxygenase is a multi-component system (Haigler & Gibson 1990 A claim for a single-component desufonative enzyme seems to have been made (Stavskaya et al 1987). The enzyme reaction was not defined, and an unverified, indirect assay was used which involved boiling the extract prior to a sulfite assay.…”

Section: Discussionmentioning

confidence: 84%

Initial steps in the degradation of benzene sulfonic acid, 4-toluene sulfonic acids, and orthanilic acid in Alcaligenes sp. strain O-1

Thurnheer¹,

Zürrer²,

Höglinger³

et al. 1990

Biodegradation

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Alcaligenes sp. strain O-1 grew with benzene sulfonate (BS) as sole carbon source for growth with either NH4 ÷ or NH4 + plus orthanilate (2-aminobenzene sulfonate, OS) as the source(s) of nitrogen. The intracellular desulfonative enzyme did not degrade 3-or 4-aminobenzene sulfonates in the medium, although the enzyme in cell extracts degraded these compounds. We deduce the presence of a selective permeability barrier to sulfonates and conclude that the first step in sulfonate metabolism is transport into the cell. Cell-free desulfonation of BS in standard reaction mixtures required 2 mol of 02 per mol. One mol of 02 was required for a catechol 2,3-dioxygenase. When meta ring cleavage was inhibited with 3-chlorocatechol in desalted extracts, about 1 mol each of 02 and of NAD(P)H per mol of BS were required for the reaction, and SO32-and catechol were recovered in high yield. Catechol was shown to be formed by dioxygenation in an experiment involving 180 2. 4-Toluene sulfonate was subject to NAD(P)H-dependent dioxygenation to yield SO32-and 4-methylcatechol, which was subject to meta cleavage. OS also required 2 mol of O2 per mol and NAD(P)H for degradation, and SO32-and NH4 + were recovered quantitatively. Inhibition of ring cleavage with 3-chlorocatechol reduced the oxygen requirement to 1 mol per mol of OS SO32-(1 mol) and an unidentified organic intermediate, but no NH4 +, were observed.

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“…This reductase component oxidized NAD(P)H and concomitantly reduced cytochrome c in vitro; hence, it was designated Ccr (cytochrome c reductase component). The reductases of several well-characterized Rieske non-haem iron oxygenases are also known to reduce cytochrome c in vitro (Subramanian et al, 1979;Haigler & Gibson, 1990b;Yu et al, 2007). Partially purified Ccr was used for the purification of the Ndm component, and the Ccr requirement for N-demethylation by Ndm was specific.…”

Section: Discussionmentioning

confidence: 99%

Characterization of a broad-specificity non-haem iron N-demethylase from Pseudomonas putida CBB5 capable of utilizing several purine alkaloids as sole carbon and nitrogen source

Summers

Louie

et al. 2011

Microbiology

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N-Demethylation of many xenobiotics and naturally occurring purine alkaloids such as caffeine and theobromine is primarily catalysed in higher organisms, ranging from fungi to mammals, by the well-studied membrane-associated cytochrome P450s. In contrast, there is no well-characterized enzyme for N-demethylation of purine alkaloids from bacteria, despite several reports on their utilization as sole source of carbon and nitrogen. Here, we provide what we believe to be the first detailed characterization of a purified N-demethylase from Pseudomonas putida CBB5. The soluble N-demethylase holoenzyme is composed of two components, a reductase component with cytochrome c reductase activity (Ccr) and a two-subunit N-demethylase component (Ndm). Ndm, with a native molecular mass of 240 kDa, is composed of NdmA (40 kDa) and NdmB (35 kDa). Ccr transfers reducing equivalents from NAD(P)H to Ndm, which catalyses an oxygen-dependent N-demethylation of methylxanthines to xanthine, formaldehyde and water. Paraxanthine and 7-methylxanthine were determined to be the best substrates, with apparent K m and kcat values of 50.4±6.8 μM and 16.2±0.6 min−1, and 63.8±7.5 μM and 94.8±3.0 min−1, respectively. Ndm also displayed activity towards caffeine, theobromine, theophylline and 3-methylxanthine, all of which are growth substrates for this organism. Ndm was deduced to be a Rieske [2Fe–2S]-domain-containing non-haem iron oxygenase based on (i) its distinct absorption spectrum and (ii) significant identity of the N-terminal sequences of NdmA and NdmB with the gene product of an uncharacterized caffeine demethylase in P. putida IF-3 and a hypothetical protein in Janthinobacterium sp. Marseille, both predicted to be Rieske non-haem iron oxygenases.

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Purification and properties of NADH-ferredoxinNAP reductase, a component of naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816

Cited by 152 publications

References 43 publications

Single Turnover Chemistry and Regulation of O2Activation by the Oxygenase Component of Naphthalene 1,2-Dioxygenase

Single Turnover Chemistry and Regulation of O2Activation by the Oxygenase Component of Naphthalene 1,2-Dioxygenase

Initial steps in the degradation of benzene sulfonic acid, 4-toluene sulfonic acids, and orthanilic acid in Alcaligenes sp. strain O-1

Characterization of a broad-specificity non-haem iron N-demethylase from Pseudomonas putida CBB5 capable of utilizing several purine alkaloids as sole carbon and nitrogen source

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