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Redox proteins of hydroxylating bacterial dioxygenases establish a regulatory cascade that prevents gratuitous induction of tetralin biodegradation genes.

Ledesma-García L, Sánchez-Azqueta A, Medina M, Reyes-Ramírez F, Santero E - Sci Rep (2016)

Bottom Line: It consists of a ferredoxin reductase (ThnA4), a ferredoxin (ThnA3) and a oxygenase (ThnA1/ThnA2), forming a NAD(P)H-ThnA4-ThnA3-ThnA1/ThnA2 electron transport chain.Here we analyze electron transfer among ThnA4, ThnA3 and ThnY by using stopped-flow spectrophotometry and determination of midpoint reduction potentials.Our results indicate that when accumulated in its reduced form ThnA3 is able to fully reduce ThnY.

View Article: PubMed Central - PubMed

Affiliation: Centro Andaluz de Biología del Desarrollo, Universidad Pablo de Olavide/Consejo Superior de Investigaciones Científicas/Junta de Andalucía, and Departamento de Biología Molecular e Ingeniería Bioquímica, Seville, Spain.

ABSTRACT
Bacterial dioxygenase systems are multicomponent enzymes that catalyze the initial degradation of many environmentally hazardous compounds. In Sphingopyxis granuli strain TFA tetralin dioxygenase hydroxylates tetralin, an organic contaminant. It consists of a ferredoxin reductase (ThnA4), a ferredoxin (ThnA3) and a oxygenase (ThnA1/ThnA2), forming a NAD(P)H-ThnA4-ThnA3-ThnA1/ThnA2 electron transport chain. ThnA3 has also a regulatory function since it prevents expression of tetralin degradation genes (thn) in the presence of non-metabolizable substrates of the catabolic pathway. This role is of physiological relevance since avoids gratuitous and wasteful production of catabolic enzymes. Our hypothesis for thn regulation implies that ThnA3 exerts its action by diverting electrons towards the regulator ThnY, an iron-sulfur flavoprotein that together with the transcriptional activator ThnR is necessary for thn gene expression. Here we analyze electron transfer among ThnA4, ThnA3 and ThnY by using stopped-flow spectrophotometry and determination of midpoint reduction potentials. Our results indicate that when accumulated in its reduced form ThnA3 is able to fully reduce ThnY. In addition, we have reproduced in vitro the regulatory circuit in the proposed physiological direction, NAD(P)H-ThnA4-ThnA3-ThnY. ThnA3 represents an unprecedented way of communication between a catabolic pathway and its regulatory system to prevent gratuitous induction.

No MeSH data available.


Related in: MedlinePlus

Anaerobic reduction of ThnA3ox by ThnA4red.(a) Spectral evolution of the reaction of ThnA3ox (~14 μM holoenzyme) with ThnA4red (~5 μM, previously reduced with 50 μM NADH) as measured by stopped-flow spectroscopy under anaerobic conditions. Spectra recorded at 0.00384 (dashed line), 0.03968, 0.2138, 0.5082, 22.14, and 220.8 s after mixing are shown. The inset shows the evolution of the absorbance at 452 nm (grey bold line) and 462 nm (thin black line), as well as their corresponding global fits (bold lines) to a three-steps model, A → B → C → D. (b) Spectroscopic properties of the intermediate pre-steady-state species. The inset shows the evolution of the obtained spectral species over the time. Species A, B, C and D are shown as continuous black thin, black bold, grey bold and grey dashed lines, respectively. Experimental conditions as in Fig. 1.
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f3: Anaerobic reduction of ThnA3ox by ThnA4red.(a) Spectral evolution of the reaction of ThnA3ox (~14 μM holoenzyme) with ThnA4red (~5 μM, previously reduced with 50 μM NADH) as measured by stopped-flow spectroscopy under anaerobic conditions. Spectra recorded at 0.00384 (dashed line), 0.03968, 0.2138, 0.5082, 22.14, and 220.8 s after mixing are shown. The inset shows the evolution of the absorbance at 452 nm (grey bold line) and 462 nm (thin black line), as well as their corresponding global fits (bold lines) to a three-steps model, A → B → C → D. (b) Spectroscopic properties of the intermediate pre-steady-state species. The inset shows the evolution of the obtained spectral species over the time. Species A, B, C and D are shown as continuous black thin, black bold, grey bold and grey dashed lines, respectively. Experimental conditions as in Fig. 1.

Mentions: Once demonstrated that ThnA4 is functionally reduced by NAD(P)H, we also analyzed its ability to transfer electrons from NADH to ThnA3ox. With this aim we followed the spectral evolution upon mixing under anaerobic conditions an excess of ThnA3ox with ThnA4red, which was formed by previous incubation of ThnA4ox with NADH (Fig. 3). The spectral shape of ThnA3ox rapidly changed after mixing with ThnA4red and its absorption peaks were displaced to 435 and 522 nm (Fig. 3a). These absorbance maxima are characteristic of reduced Rieske-type ferredoxins of aromatic systems such as BphA3 of the biphenyl dioxygenase complex from Pseudomonas sp. KKS10221, the ferredoxinNAP component of naphthalene dioxygenase from Pseudomonas17 and the CarAc component of the carbazole 1,9α-dioxygenase15. The overall process fit to a three-step model (Fig. 3a, inset, and 3b). Species A resembled the ThnA3ox spectrum. Transition of A into B was related with a slight increment in the absorption of the 460 band and a decrease in the 600 nm band, changes consistent with the production of the interaction between both proteins. Transition of B into C related with absorption decrease in the whole wavelength range consistent with reduction of ThnA3ox by the NADH reduced ThnA4. Transformation of species C into D is a considerably slower process that accounts for a very small change in amplitude, probably related with final consumption of the excess of reduced coenzyme and the achievement of the steady-state equilibrium.


Redox proteins of hydroxylating bacterial dioxygenases establish a regulatory cascade that prevents gratuitous induction of tetralin biodegradation genes.

Ledesma-García L, Sánchez-Azqueta A, Medina M, Reyes-Ramírez F, Santero E - Sci Rep (2016)

Anaerobic reduction of ThnA3ox by ThnA4red.(a) Spectral evolution of the reaction of ThnA3ox (~14 μM holoenzyme) with ThnA4red (~5 μM, previously reduced with 50 μM NADH) as measured by stopped-flow spectroscopy under anaerobic conditions. Spectra recorded at 0.00384 (dashed line), 0.03968, 0.2138, 0.5082, 22.14, and 220.8 s after mixing are shown. The inset shows the evolution of the absorbance at 452 nm (grey bold line) and 462 nm (thin black line), as well as their corresponding global fits (bold lines) to a three-steps model, A → B → C → D. (b) Spectroscopic properties of the intermediate pre-steady-state species. The inset shows the evolution of the obtained spectral species over the time. Species A, B, C and D are shown as continuous black thin, black bold, grey bold and grey dashed lines, respectively. Experimental conditions as in Fig. 1.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4814904&req=5

f3: Anaerobic reduction of ThnA3ox by ThnA4red.(a) Spectral evolution of the reaction of ThnA3ox (~14 μM holoenzyme) with ThnA4red (~5 μM, previously reduced with 50 μM NADH) as measured by stopped-flow spectroscopy under anaerobic conditions. Spectra recorded at 0.00384 (dashed line), 0.03968, 0.2138, 0.5082, 22.14, and 220.8 s after mixing are shown. The inset shows the evolution of the absorbance at 452 nm (grey bold line) and 462 nm (thin black line), as well as their corresponding global fits (bold lines) to a three-steps model, A → B → C → D. (b) Spectroscopic properties of the intermediate pre-steady-state species. The inset shows the evolution of the obtained spectral species over the time. Species A, B, C and D are shown as continuous black thin, black bold, grey bold and grey dashed lines, respectively. Experimental conditions as in Fig. 1.
Mentions: Once demonstrated that ThnA4 is functionally reduced by NAD(P)H, we also analyzed its ability to transfer electrons from NADH to ThnA3ox. With this aim we followed the spectral evolution upon mixing under anaerobic conditions an excess of ThnA3ox with ThnA4red, which was formed by previous incubation of ThnA4ox with NADH (Fig. 3). The spectral shape of ThnA3ox rapidly changed after mixing with ThnA4red and its absorption peaks were displaced to 435 and 522 nm (Fig. 3a). These absorbance maxima are characteristic of reduced Rieske-type ferredoxins of aromatic systems such as BphA3 of the biphenyl dioxygenase complex from Pseudomonas sp. KKS10221, the ferredoxinNAP component of naphthalene dioxygenase from Pseudomonas17 and the CarAc component of the carbazole 1,9α-dioxygenase15. The overall process fit to a three-step model (Fig. 3a, inset, and 3b). Species A resembled the ThnA3ox spectrum. Transition of A into B was related with a slight increment in the absorption of the 460 band and a decrease in the 600 nm band, changes consistent with the production of the interaction between both proteins. Transition of B into C related with absorption decrease in the whole wavelength range consistent with reduction of ThnA3ox by the NADH reduced ThnA4. Transformation of species C into D is a considerably slower process that accounts for a very small change in amplitude, probably related with final consumption of the excess of reduced coenzyme and the achievement of the steady-state equilibrium.

Bottom Line: It consists of a ferredoxin reductase (ThnA4), a ferredoxin (ThnA3) and a oxygenase (ThnA1/ThnA2), forming a NAD(P)H-ThnA4-ThnA3-ThnA1/ThnA2 electron transport chain.Here we analyze electron transfer among ThnA4, ThnA3 and ThnY by using stopped-flow spectrophotometry and determination of midpoint reduction potentials.Our results indicate that when accumulated in its reduced form ThnA3 is able to fully reduce ThnY.

View Article: PubMed Central - PubMed

Affiliation: Centro Andaluz de Biología del Desarrollo, Universidad Pablo de Olavide/Consejo Superior de Investigaciones Científicas/Junta de Andalucía, and Departamento de Biología Molecular e Ingeniería Bioquímica, Seville, Spain.

ABSTRACT
Bacterial dioxygenase systems are multicomponent enzymes that catalyze the initial degradation of many environmentally hazardous compounds. In Sphingopyxis granuli strain TFA tetralin dioxygenase hydroxylates tetralin, an organic contaminant. It consists of a ferredoxin reductase (ThnA4), a ferredoxin (ThnA3) and a oxygenase (ThnA1/ThnA2), forming a NAD(P)H-ThnA4-ThnA3-ThnA1/ThnA2 electron transport chain. ThnA3 has also a regulatory function since it prevents expression of tetralin degradation genes (thn) in the presence of non-metabolizable substrates of the catabolic pathway. This role is of physiological relevance since avoids gratuitous and wasteful production of catabolic enzymes. Our hypothesis for thn regulation implies that ThnA3 exerts its action by diverting electrons towards the regulator ThnY, an iron-sulfur flavoprotein that together with the transcriptional activator ThnR is necessary for thn gene expression. Here we analyze electron transfer among ThnA4, ThnA3 and ThnY by using stopped-flow spectrophotometry and determination of midpoint reduction potentials. Our results indicate that when accumulated in its reduced form ThnA3 is able to fully reduce ThnY. In addition, we have reproduced in vitro the regulatory circuit in the proposed physiological direction, NAD(P)H-ThnA4-ThnA3-ThnY. ThnA3 represents an unprecedented way of communication between a catabolic pathway and its regulatory system to prevent gratuitous induction.

No MeSH data available.


Related in: MedlinePlus