Toluene dioxygenase: Purification of an iron-sulfur protein by affinity chromatography

Subramanian, V.; Liu, Te-Ning; Yeh, W K; Gibson, David T.

doi:10.1016/0006-291x(79)91998-3

Cited by 97 publications

(73 citation statements)

References 14 publications

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“…2b). Exogenous addition of 50 mM Fe 2+ to the Ndm enzyme reaction mixture stimulated Ndm enzyme activity, similar to other other Rieske [2Fe-2S]-domain-containing non-haem iron oxygenases such as toluene, naphthalene and biphenyl dioxygenases (Ensley & Gibson, 1983;Subramanian et al, 1979;Yu et al, 2007). Generally, the mononuclear ferrous iron in Rieske oxygenases is not tightly bound and often dissociates during protein purification.…”

Section: Discussionsupporting

confidence: 77%

“…9816 naphthalene dioxygenase system (Haigler & Gibson, 1990a, b) did not support the N-demethylase reaction by Ndm. In contrast, the reductase components of toluene dioxygenase of P. putida (Subramanian et al, 1979) and biphenyl dioxygenase of Sphingobium yanoikuyae B1 (Yu et al, 2007) are easily substituted by spinach ferredoxin reductase and the reductase of naphthalene dioxygenase, respectively. A proposed reaction scheme for N-demethylation of paraxanthine (and other methylxanthines) by Ccr plus Ndm is presented in Fig.…”

Section: Discussionmentioning

confidence: 95%

“…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%

“…strain 9816 (Haigler & Gibson, 1990a, b), 30 and 100 mg of the respective proteins were used. Spinach ferredoxin reductase and ferredoxin were also substituted for Ccr as described by Subramanian et al (1979).…”

Section: Methodsmentioning

confidence: 99%

“…Ndm is predicted to be a Rieske [2Fe-2S]-domain-containing nonhaem iron oxygenase, and oxygenases of this type are known to be promiscuous in terms of the partner reductases (Subramanian et al, 1979;Yu et al, 2007). However, Ndm did not function in the presence of the reductase and ferredoxin components of Pseudomonas sp.…”

Section: Reductase Requirement Of Ndmmentioning

confidence: 99%

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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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Section: Discussionsupporting

confidence: 77%

Section: Discussionmentioning

confidence: 95%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Reductase Requirement Of Ndmmentioning

confidence: 99%

See 3 more Smart Citations

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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Oxygenase Enzymes: Role in Biodegradation

Haddock

2003

Encyclopedia of Environmental Microbiology

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Production of toluene cis‐glycol by Pseudomonas putida in glucose feb‐batch culture

Jenkins¹,

Stephens²,

Dalton³

1987

Biotech & Bioengineering

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Toluene was oxidized by a mutant strain of Pseudomonas putida (strain NG1) to toluene Cis-Glycol (TCG). Product was accumulated in fed-batch cultures to concentrations (18-24 g/L) higher than hitherto achieved. In vitro activities of toluene dioxygenase from P. Putida NG1 were fivefold lower than that from the toluene-grown wild-type organism, whereas comparable activities of both catechol 2,3- and catechol 1,2-oxygenase were obtained; irreversible inhibition of toluene dioxygenase activity by TCG was shown in vitro. Ammonia deprivation during the production phase limited the growth of revertant organisms but had little effect on either the duration (25h) of the process or the final concentration of TCG achieved. The rate of glucose utilization decreased throughout the biotransformation and cell death accompanied the cessation of TCG accumulation in cultures. These changes were a consequence of TCG formation and a cooperative toxic effect was demonstrated for toluene and TCG. Adenylate energy charge values decreased from ca. 0.8 to 0.2 over the course of the biotransformation but were maintained above 0.5 in the absence of TCG. Similarly, cellular AMP levels increased dramatically during biotransformation, presumably as a consequence of RNA degradation, but were maintained at low levels in the absence of TCG. The results suggest that TCG is the mediate of a gradual deterioration in the state of the culture which leads to a loss of both in vivo and in vitro toluence dioxygenase activity and a marked decrease in culture viability.

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Toluene dioxygenase: Purification of an iron-sulfur protein by affinity chromatography

Cited by 97 publications

References 14 publications

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

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

Oxygenase Enzymes: Role in Biodegradation

Production of toluene cis‐glycol by Pseudomonas putida in glucose feb‐batch culture

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