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Functional characterization of diverse ring-hydroxylating oxygenases and induction of complex aromatic catabolic gene clusters in Sphingobium sp. PNB.

Khara P, Roy M, Chakraborty J, Ghosal D, Dutta TK - FEBS Open Bio (2014)

Bottom Line: Comparison of the map of the catabolic genes with that of different sphingomonads revealed a similar arrangement of gene clusters that harbors seven sets of RHO terminal components and a sole set of electron transport (ET) proteins.The presence of distinctly conserved amino acid residues in ferredoxin and in silico molecular docking analyses of ferredoxin with the well characterized terminal oxygenase components indicated the structural uniqueness of the ET component in sphingomonads.The RHO AhdA1bA2b was functionally characterized for the first time and was found to be capable of transforming ethylbenzene, propylbenzene, cumene, p-cymene and biphenyl, in addition to a number of polycyclic aromatic hydrocarbons.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Bose Institute, P-1/12 C.I.T. Scheme VII M, Kolkata 700054, India.

ABSTRACT
Sphingobium sp. PNB, like other sphingomonads, has multiple ring-hydroxylating oxygenase (RHO) genes. Three different fosmid clones have been sequenced to identify the putative genes responsible for the degradation of various aromatics in this bacterial strain. Comparison of the map of the catabolic genes with that of different sphingomonads revealed a similar arrangement of gene clusters that harbors seven sets of RHO terminal components and a sole set of electron transport (ET) proteins. The presence of distinctly conserved amino acid residues in ferredoxin and in silico molecular docking analyses of ferredoxin with the well characterized terminal oxygenase components indicated the structural uniqueness of the ET component in sphingomonads. The predicted substrate specificities, derived from the phylogenetic relationship of each of the RHOs, were examined based on transformation of putative substrates and their structural homologs by the recombinant strains expressing each of the oxygenases and the sole set of available ET proteins. The RHO AhdA1bA2b was functionally characterized for the first time and was found to be capable of transforming ethylbenzene, propylbenzene, cumene, p-cymene and biphenyl, in addition to a number of polycyclic aromatic hydrocarbons. Overexpression of aromatic catabolic genes in strain PNB, revealed by real-time PCR analyses, is a way forward to understand the complex regulation of degradative genes in sphingomonads.

No MeSH data available.


Related in: MedlinePlus

Molecular docking of oxygenase–ferredoxin complexes. The surface plots (side view) of the docked complexes of respective ferredoxin and terminal oxygenase components of (A) naphthalene 1,2-dioxygenase from Pseudomonas putida NCIB 9816-4, (B) biphenyl 2,3-dioxygenase from Sphingobium yanoikuyae B1 and (C) terminal oxygenase AhdA1fA2f from Sphingobium sp. PNB. In each structure, the visible α-subunits are colored in light green and blue, while the visible β-subunits are shown in dark green and slate. The ferredoxins in each complex are colored pink. Black dotted circle in each complex shows the region where the Rieske clusters of ferredoxin and oxygenase large subunit lie in close proximity for electron transport, while the same as enlarged (D, E and F) are shown in the corresponding cartoon representations. Distance between each pair of redox centre is shown in black dotted lines. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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f0020: Molecular docking of oxygenase–ferredoxin complexes. The surface plots (side view) of the docked complexes of respective ferredoxin and terminal oxygenase components of (A) naphthalene 1,2-dioxygenase from Pseudomonas putida NCIB 9816-4, (B) biphenyl 2,3-dioxygenase from Sphingobium yanoikuyae B1 and (C) terminal oxygenase AhdA1fA2f from Sphingobium sp. PNB. In each structure, the visible α-subunits are colored in light green and blue, while the visible β-subunits are shown in dark green and slate. The ferredoxins in each complex are colored pink. Black dotted circle in each complex shows the region where the Rieske clusters of ferredoxin and oxygenase large subunit lie in close proximity for electron transport, while the same as enlarged (D, E and F) are shown in the corresponding cartoon representations. Distance between each pair of redox centre is shown in black dotted lines. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Mentions: Docking experiments were performed using GRAMM-X to assess the interactions between three different oxygenase–ferredoxin complexes, viz. NDO-O98164:NDO-F98164, BDO-OB1:BDO-FB1 and AHD-OPNB:AHD-FPNB. Out of 50 docked complex models, ranked according to the scoring function, one showing distance between the Rieske clusters of ferredoxin and oxygenase α-subunit closest to 14 Å threshold [25] was considered as the best-fit model (Fig. 4). The observed distances were 16, 16.3 and 14.1 Å respectively for NDO-O98164:NDO-F98164, BDO-OB1:BDO-FB1 and AHD-OPNB:AHD-FPNB complexes. For each docked complex, the interface residues of both ferredoxin and oxygenase α-subunit are shown in Table S6. As observed from the docked poses, NDO-F98164 binds at the depression between two adjacent α-subunits of NDO-O98164, which is in congruence with an earlier study [26]. On the contrary, both BDO-FB1 and AHD-FPNB seem to bind at a pronounced depression formed by two α- and two β-subunits (Fig. 4), the other putative ferredoxin binding site, as postulated by Ashikawa et al.[26]. Mapping of predicted interface residues onto the alignment of ferredoxin sequences obtained from various xenobiotic degrading organisms depicted a few differentially conserved amino acid residues (Phe66, Gly68 and Phe81) in sphingomonads (Fig. 3B). Thus, it is believed that the Rieske-type [2Fe–2S] ferredoxins of sphingomonads might have evolved to complement multiple oxygenases present in these organisms.


Functional characterization of diverse ring-hydroxylating oxygenases and induction of complex aromatic catabolic gene clusters in Sphingobium sp. PNB.

Khara P, Roy M, Chakraborty J, Ghosal D, Dutta TK - FEBS Open Bio (2014)

Molecular docking of oxygenase–ferredoxin complexes. The surface plots (side view) of the docked complexes of respective ferredoxin and terminal oxygenase components of (A) naphthalene 1,2-dioxygenase from Pseudomonas putida NCIB 9816-4, (B) biphenyl 2,3-dioxygenase from Sphingobium yanoikuyae B1 and (C) terminal oxygenase AhdA1fA2f from Sphingobium sp. PNB. In each structure, the visible α-subunits are colored in light green and blue, while the visible β-subunits are shown in dark green and slate. The ferredoxins in each complex are colored pink. Black dotted circle in each complex shows the region where the Rieske clusters of ferredoxin and oxygenase large subunit lie in close proximity for electron transport, while the same as enlarged (D, E and F) are shown in the corresponding cartoon representations. Distance between each pair of redox centre is shown in black dotted lines. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
© Copyright Policy - CC BY
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4048848&req=5

f0020: Molecular docking of oxygenase–ferredoxin complexes. The surface plots (side view) of the docked complexes of respective ferredoxin and terminal oxygenase components of (A) naphthalene 1,2-dioxygenase from Pseudomonas putida NCIB 9816-4, (B) biphenyl 2,3-dioxygenase from Sphingobium yanoikuyae B1 and (C) terminal oxygenase AhdA1fA2f from Sphingobium sp. PNB. In each structure, the visible α-subunits are colored in light green and blue, while the visible β-subunits are shown in dark green and slate. The ferredoxins in each complex are colored pink. Black dotted circle in each complex shows the region where the Rieske clusters of ferredoxin and oxygenase large subunit lie in close proximity for electron transport, while the same as enlarged (D, E and F) are shown in the corresponding cartoon representations. Distance between each pair of redox centre is shown in black dotted lines. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Mentions: Docking experiments were performed using GRAMM-X to assess the interactions between three different oxygenase–ferredoxin complexes, viz. NDO-O98164:NDO-F98164, BDO-OB1:BDO-FB1 and AHD-OPNB:AHD-FPNB. Out of 50 docked complex models, ranked according to the scoring function, one showing distance between the Rieske clusters of ferredoxin and oxygenase α-subunit closest to 14 Å threshold [25] was considered as the best-fit model (Fig. 4). The observed distances were 16, 16.3 and 14.1 Å respectively for NDO-O98164:NDO-F98164, BDO-OB1:BDO-FB1 and AHD-OPNB:AHD-FPNB complexes. For each docked complex, the interface residues of both ferredoxin and oxygenase α-subunit are shown in Table S6. As observed from the docked poses, NDO-F98164 binds at the depression between two adjacent α-subunits of NDO-O98164, which is in congruence with an earlier study [26]. On the contrary, both BDO-FB1 and AHD-FPNB seem to bind at a pronounced depression formed by two α- and two β-subunits (Fig. 4), the other putative ferredoxin binding site, as postulated by Ashikawa et al.[26]. Mapping of predicted interface residues onto the alignment of ferredoxin sequences obtained from various xenobiotic degrading organisms depicted a few differentially conserved amino acid residues (Phe66, Gly68 and Phe81) in sphingomonads (Fig. 3B). Thus, it is believed that the Rieske-type [2Fe–2S] ferredoxins of sphingomonads might have evolved to complement multiple oxygenases present in these organisms.

Bottom Line: Comparison of the map of the catabolic genes with that of different sphingomonads revealed a similar arrangement of gene clusters that harbors seven sets of RHO terminal components and a sole set of electron transport (ET) proteins.The presence of distinctly conserved amino acid residues in ferredoxin and in silico molecular docking analyses of ferredoxin with the well characterized terminal oxygenase components indicated the structural uniqueness of the ET component in sphingomonads.The RHO AhdA1bA2b was functionally characterized for the first time and was found to be capable of transforming ethylbenzene, propylbenzene, cumene, p-cymene and biphenyl, in addition to a number of polycyclic aromatic hydrocarbons.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Bose Institute, P-1/12 C.I.T. Scheme VII M, Kolkata 700054, India.

ABSTRACT
Sphingobium sp. PNB, like other sphingomonads, has multiple ring-hydroxylating oxygenase (RHO) genes. Three different fosmid clones have been sequenced to identify the putative genes responsible for the degradation of various aromatics in this bacterial strain. Comparison of the map of the catabolic genes with that of different sphingomonads revealed a similar arrangement of gene clusters that harbors seven sets of RHO terminal components and a sole set of electron transport (ET) proteins. The presence of distinctly conserved amino acid residues in ferredoxin and in silico molecular docking analyses of ferredoxin with the well characterized terminal oxygenase components indicated the structural uniqueness of the ET component in sphingomonads. The predicted substrate specificities, derived from the phylogenetic relationship of each of the RHOs, were examined based on transformation of putative substrates and their structural homologs by the recombinant strains expressing each of the oxygenases and the sole set of available ET proteins. The RHO AhdA1bA2b was functionally characterized for the first time and was found to be capable of transforming ethylbenzene, propylbenzene, cumene, p-cymene and biphenyl, in addition to a number of polycyclic aromatic hydrocarbons. Overexpression of aromatic catabolic genes in strain PNB, revealed by real-time PCR analyses, is a way forward to understand the complex regulation of degradative genes in sphingomonads.

No MeSH data available.


Related in: MedlinePlus