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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

(A) Neighbor-joining tree and (B) sequence alignment of Rieske type [2Fe–2S] ferredoxins from different well studied xenobiotic degrading bacteria. In the tree, values at each node indicate level of bootstrap support based on 100 resampled datasets while bootstrap values below 50% are not shown. An unrelated ferredoxin (PhtAc) from Mycobacterium vanbaalenii PYR-1 (GenBank: AAQ91918) was used as outgroup and position of the root has been indicated by an arrow. Bar represents 0.05 substitutions per amino acid. Identical and similar residues in the sequence alignment are shaded in black and grey, respectively.
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f0015: (A) Neighbor-joining tree and (B) sequence alignment of Rieske type [2Fe–2S] ferredoxins from different well studied xenobiotic degrading bacteria. In the tree, values at each node indicate level of bootstrap support based on 100 resampled datasets while bootstrap values below 50% are not shown. An unrelated ferredoxin (PhtAc) from Mycobacterium vanbaalenii PYR-1 (GenBank: AAQ91918) was used as outgroup and position of the root has been indicated by an arrow. Bar represents 0.05 substitutions per amino acid. Identical and similar residues in the sequence alignment are shaded in black and grey, respectively.

Mentions: As in other sphingomonads, only one ferredoxin and one ferredoxin reductase were identified, each encoded in a separate cluster [3]. Analysis of AhdA3 revealed it to be a Rieske [2Fe–2S] type ferredoxin with the conserved C-X-H-Xn-C-X2-H motif, a distinct feature of this family [24]. Fig. 3A illustrates the phylogenetic relation of AhdA3 with those of homologous sequences from various xenobiotic degrading organisms described in Table S4. Although these proteins share a conserved domain, AhdA3 shares limited sequence homology (<50% identity) with its non-sphingomonad counterparts. It is evident from the dendogram that the ferredoxins of the sphingomonads are highly similar (81–100% identity at amino acid level) and cluster together. It may be mentioned that AhdA3 and homologous sequences in sphingomonads displayed a few differentially conserved amino acids across the length of the proteins (Met26, Asn33, Gln57, Ile61, Phe66, Gly68, Ser70, Ala77, Ala80 and Phe81) in comparison to that of non-sphingomonads (Fig. 3B). However, the ferredoxins from few non-sphingomonads, viz. EtbAc, PhnA3 and PhnAb, complementing ethylbenzene dioxygenase in Rhodococcus jostii RHA1, PAH dioxygenase in Cycloclasticus sp. A5 and phenanthrene dioxygenase in Alcaligenes faecalis AFK2, respectively, were found to be phylogenetically close to the sphingomonad ferredoxins with significant sequence similarity (Fig. 3). This observation was in congruence with the phylogenetic relatedness of corresponding oxygenase from these organisms to those of the sphingomonads (Fig. 2), indicating a possible event of lateral transfer of oxygenase gene clusters among them.


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)

(A) Neighbor-joining tree and (B) sequence alignment of Rieske type [2Fe–2S] ferredoxins from different well studied xenobiotic degrading bacteria. In the tree, values at each node indicate level of bootstrap support based on 100 resampled datasets while bootstrap values below 50% are not shown. An unrelated ferredoxin (PhtAc) from Mycobacterium vanbaalenii PYR-1 (GenBank: AAQ91918) was used as outgroup and position of the root has been indicated by an arrow. Bar represents 0.05 substitutions per amino acid. Identical and similar residues in the sequence alignment are shaded in black and grey, respectively.
© Copyright Policy - CC BY
Related In: Results  -  Collection

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

f0015: (A) Neighbor-joining tree and (B) sequence alignment of Rieske type [2Fe–2S] ferredoxins from different well studied xenobiotic degrading bacteria. In the tree, values at each node indicate level of bootstrap support based on 100 resampled datasets while bootstrap values below 50% are not shown. An unrelated ferredoxin (PhtAc) from Mycobacterium vanbaalenii PYR-1 (GenBank: AAQ91918) was used as outgroup and position of the root has been indicated by an arrow. Bar represents 0.05 substitutions per amino acid. Identical and similar residues in the sequence alignment are shaded in black and grey, respectively.
Mentions: As in other sphingomonads, only one ferredoxin and one ferredoxin reductase were identified, each encoded in a separate cluster [3]. Analysis of AhdA3 revealed it to be a Rieske [2Fe–2S] type ferredoxin with the conserved C-X-H-Xn-C-X2-H motif, a distinct feature of this family [24]. Fig. 3A illustrates the phylogenetic relation of AhdA3 with those of homologous sequences from various xenobiotic degrading organisms described in Table S4. Although these proteins share a conserved domain, AhdA3 shares limited sequence homology (<50% identity) with its non-sphingomonad counterparts. It is evident from the dendogram that the ferredoxins of the sphingomonads are highly similar (81–100% identity at amino acid level) and cluster together. It may be mentioned that AhdA3 and homologous sequences in sphingomonads displayed a few differentially conserved amino acids across the length of the proteins (Met26, Asn33, Gln57, Ile61, Phe66, Gly68, Ser70, Ala77, Ala80 and Phe81) in comparison to that of non-sphingomonads (Fig. 3B). However, the ferredoxins from few non-sphingomonads, viz. EtbAc, PhnA3 and PhnAb, complementing ethylbenzene dioxygenase in Rhodococcus jostii RHA1, PAH dioxygenase in Cycloclasticus sp. A5 and phenanthrene dioxygenase in Alcaligenes faecalis AFK2, respectively, were found to be phylogenetically close to the sphingomonad ferredoxins with significant sequence similarity (Fig. 3). This observation was in congruence with the phylogenetic relatedness of corresponding oxygenase from these organisms to those of the sphingomonads (Fig. 2), indicating a possible event of lateral transfer of oxygenase gene clusters among them.

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