Limits...
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 ThnYox by ThnA3red.(a) Spectral evolution of the reaction of ThnYox (~8 μM) with photoreduced ThnA3red (~24 μM holoenzyme) as measured by stopped-flow spectrophotometry. Spectra recorded at 0.00128, 0.04736, 0.4109, 0.8614, 1.778, 3.488, 5.382, 8.25, 11.74, 17.8, and 54.38 s after mixing are shown. Direction of the spectral evolution is indicated by arrows. The inset shows the absorbance evolution at 451 nm (black line) and 531 nm (grey line) and their corresponding global fits to a two-steps model, A → B → C (bold black lines). (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 and C are shown as continuous black thin, black bold and grey bold lines, respectively. Measurements carried out in potassium phosphate 50 mM, pH 7.4, NaCl 10 mM, glycerine 5%.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4814904&req=5

f1: Anaerobic reduction of ThnYox by ThnA3red.(a) Spectral evolution of the reaction of ThnYox (~8 μM) with photoreduced ThnA3red (~24 μM holoenzyme) as measured by stopped-flow spectrophotometry. Spectra recorded at 0.00128, 0.04736, 0.4109, 0.8614, 1.778, 3.488, 5.382, 8.25, 11.74, 17.8, and 54.38 s after mixing are shown. Direction of the spectral evolution is indicated by arrows. The inset shows the absorbance evolution at 451 nm (black line) and 531 nm (grey line) and their corresponding global fits to a two-steps model, A → B → C (bold black lines). (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 and C are shown as continuous black thin, black bold and grey bold lines, respectively. Measurements carried out in potassium phosphate 50 mM, pH 7.4, NaCl 10 mM, glycerine 5%.

Mentions: To investigate these facts, the hexahistidine-tagged versions of each of these proteins were purified by using metal chelate affinity and size exclusion chromatography. The His6-ThnY holoenzyme, containing FAD and a plant-type [2Fe-2S] cluster, was purified as previously reported9. ThnA3-His6 was here purified for the first time as a protein with an apparent molecular mass of 14 kDa, calculated from its mobility in SDS-PAGE, which agrees with the calculated from its coding sequence. The ThnA3 sequence bears the highly conserved metal-binding motif, CXHX15–17CX2H, containing the two cysteines and two histidines that co-ordinate the Rieske-type [2Fe-2S] cluster. Solutions of purified ThnA3ox were brown-coloured and have the typical absorption spectrum of a Rieske-type [2Fe-2S] cluster with maxima at 280, 320, and 461 nm, and a shoulder at 580 nm, and became fully reduced by dithionite (Supplementary Fig. S1a). Hence, ThnA3 displays similar spectral properties to those of the related ferredoxins from benzene, toluene, biphenyl, carbazole and napthalene dioxygenase systems12. Nevertheless, cluster incorporation in recombinant ThnA3 was incomplete, with an average of 35% of [2Fe-2S] incorporation, an observation commonly associated with over-expressed iron-sulfur proteins13. Herein this percentage was considered to calculate the amount of holo-ThnA3 in our kinetic and potentiometric assays. The electron transfer process from ThnA3red to ThnYox was analyzed by using stopped-flow spectroscopy, a method that can provide information on specific steps in the electron transfer reaction sequence and, therefore, will only imply ThnA3 in its holoprotein form when observing electron transfer. To do that we mixed a ~4-fold excess of an in vitro photoreduced solution of ThnA3 with ThnYox, and evolution of the process was followed over the visible range observing a general decrease in the absorbance. As shown in Fig. 1a, the excess of ThnA3red was able to fully reduce ThnYox under anaerobic conditions without detection of any traces of a semiquinone intermediate state. Different profiles for absorption evolution at 450 nm and 531 nm (inset Fig. 1a) as well as global analysis of the spectral evolution along the process were consistent with a two-step model, A → B → C, where three spectroscopic species can be distinguished (Fig. 1b). The initial species, A, practically results in the spectrum of ThnYox. Conversion of A into B occurs with an observed rate constant, kA→B, of 17.6 ± 1.5 min−1 under conditions of Fig. 1, with an absorbance decrease at the flavin band-I (around 450 nm) consistent with the two-electron reduction of the FAD cofactor of ThnY. In agreement, the spectrum of species B is consistent with the [2Fe-2S] cluster of ThnY remaining in the oxidized state. Spectroscopic changes for the final transformation of species B into C agree with the subsequent reduction of this [2Fe-2S] cluster (kB→C = 4.6 ± 0.6 min−1). Thus, spectrum for species C is consistent with a fully reduced ThnY.


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 ThnYox by ThnA3red.(a) Spectral evolution of the reaction of ThnYox (~8 μM) with photoreduced ThnA3red (~24 μM holoenzyme) as measured by stopped-flow spectrophotometry. Spectra recorded at 0.00128, 0.04736, 0.4109, 0.8614, 1.778, 3.488, 5.382, 8.25, 11.74, 17.8, and 54.38 s after mixing are shown. Direction of the spectral evolution is indicated by arrows. The inset shows the absorbance evolution at 451 nm (black line) and 531 nm (grey line) and their corresponding global fits to a two-steps model, A → B → C (bold black lines). (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 and C are shown as continuous black thin, black bold and grey bold lines, respectively. Measurements carried out in potassium phosphate 50 mM, pH 7.4, NaCl 10 mM, glycerine 5%.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: Anaerobic reduction of ThnYox by ThnA3red.(a) Spectral evolution of the reaction of ThnYox (~8 μM) with photoreduced ThnA3red (~24 μM holoenzyme) as measured by stopped-flow spectrophotometry. Spectra recorded at 0.00128, 0.04736, 0.4109, 0.8614, 1.778, 3.488, 5.382, 8.25, 11.74, 17.8, and 54.38 s after mixing are shown. Direction of the spectral evolution is indicated by arrows. The inset shows the absorbance evolution at 451 nm (black line) and 531 nm (grey line) and their corresponding global fits to a two-steps model, A → B → C (bold black lines). (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 and C are shown as continuous black thin, black bold and grey bold lines, respectively. Measurements carried out in potassium phosphate 50 mM, pH 7.4, NaCl 10 mM, glycerine 5%.
Mentions: To investigate these facts, the hexahistidine-tagged versions of each of these proteins were purified by using metal chelate affinity and size exclusion chromatography. The His6-ThnY holoenzyme, containing FAD and a plant-type [2Fe-2S] cluster, was purified as previously reported9. ThnA3-His6 was here purified for the first time as a protein with an apparent molecular mass of 14 kDa, calculated from its mobility in SDS-PAGE, which agrees with the calculated from its coding sequence. The ThnA3 sequence bears the highly conserved metal-binding motif, CXHX15–17CX2H, containing the two cysteines and two histidines that co-ordinate the Rieske-type [2Fe-2S] cluster. Solutions of purified ThnA3ox were brown-coloured and have the typical absorption spectrum of a Rieske-type [2Fe-2S] cluster with maxima at 280, 320, and 461 nm, and a shoulder at 580 nm, and became fully reduced by dithionite (Supplementary Fig. S1a). Hence, ThnA3 displays similar spectral properties to those of the related ferredoxins from benzene, toluene, biphenyl, carbazole and napthalene dioxygenase systems12. Nevertheless, cluster incorporation in recombinant ThnA3 was incomplete, with an average of 35% of [2Fe-2S] incorporation, an observation commonly associated with over-expressed iron-sulfur proteins13. Herein this percentage was considered to calculate the amount of holo-ThnA3 in our kinetic and potentiometric assays. The electron transfer process from ThnA3red to ThnYox was analyzed by using stopped-flow spectroscopy, a method that can provide information on specific steps in the electron transfer reaction sequence and, therefore, will only imply ThnA3 in its holoprotein form when observing electron transfer. To do that we mixed a ~4-fold excess of an in vitro photoreduced solution of ThnA3 with ThnYox, and evolution of the process was followed over the visible range observing a general decrease in the absorbance. As shown in Fig. 1a, the excess of ThnA3red was able to fully reduce ThnYox under anaerobic conditions without detection of any traces of a semiquinone intermediate state. Different profiles for absorption evolution at 450 nm and 531 nm (inset Fig. 1a) as well as global analysis of the spectral evolution along the process were consistent with a two-step model, A → B → C, where three spectroscopic species can be distinguished (Fig. 1b). The initial species, A, practically results in the spectrum of ThnYox. Conversion of A into B occurs with an observed rate constant, kA→B, of 17.6 ± 1.5 min−1 under conditions of Fig. 1, with an absorbance decrease at the flavin band-I (around 450 nm) consistent with the two-electron reduction of the FAD cofactor of ThnY. In agreement, the spectrum of species B is consistent with the [2Fe-2S] cluster of ThnY remaining in the oxidized state. Spectroscopic changes for the final transformation of species B into C agree with the subsequent reduction of this [2Fe-2S] cluster (kB→C = 4.6 ± 0.6 min−1). Thus, spectrum for species C is consistent with a fully reduced ThnY.

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