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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

Dendogram showing the relatedness of α-subunit of bacterial aromatic RHOs along with the known biochemical reactions catalyzed. Class A, B and C RHOs (following classification scheme as described by Chakraborty et al.[6]) are shown in shades of green, brown and blue, respectively. Each shade within a class represents different reaction chemistry (with respect to oxygenation sites) while the lightest shade in each class represents the α-subunit belonging to sphingomonads. Values at each node indicate level of bootstrap support based on 100 resampled datasets while bootstrap values below 50% are not shown. A Class D carbazole dioxygenase (CarAaI) from Sphingomonas sp. KA1 (GenBank: YP_717981) was used as outgroup. (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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f0010: Dendogram showing the relatedness of α-subunit of bacterial aromatic RHOs along with the known biochemical reactions catalyzed. Class A, B and C RHOs (following classification scheme as described by Chakraborty et al.[6]) are shown in shades of green, brown and blue, respectively. Each shade within a class represents different reaction chemistry (with respect to oxygenation sites) while the lightest shade in each class represents the α-subunit belonging to sphingomonads. Values at each node indicate level of bootstrap support based on 100 resampled datasets while bootstrap values below 50% are not shown. A Class D carbazole dioxygenase (CarAaI) from Sphingomonas sp. KA1 (GenBank: YP_717981) was used as outgroup. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Mentions: Sequence analysis revealed seven sets of putative α- and β-subunit RHO genes, with one of the α-subunits (ahdA1a) disrupted. Each α subunit contains an N-terminal ISP domain, with a conserved Rieske [2Fe–2S] center, a C-terminal catalytic domain having a conserved mononuclear iron-binding site and a conserved aspartate, which is known to facilitate inter-subunit electron transfer between ISP and catalytic domains of α-subunits [14,17,18]. The genes encoding β-subunits were found adjacent to that of the α-subunits in all the sets of oxygenases indicating possible co-evolution of α- and β-subunits and the presence of hetero-multimeric (αnβn)-type of RHOs in strain PNB. Fig. 2 illustrates the phylogenetic relation of the α-subunit protein sequences (AhdA1b, AhdA1c, AhdA1d, AhdA1e, AhdA1f and XylX) in strain PNB and the homologous sequences from other organisms, as mentioned in Table S3. Although the α-subunits in strain PNB share conserved domain regions, their nucleotide sequences and deduced amino acid sequences share limited homology with that of the nonsphingomonad counterparts. Moreover, phylogenetic analysis reveals that the individual α-subunit proteins in sphingomonads are distantly related (Fig. 2). Pairwise sequence alignments among the α-subunit in strain PNB, showed identities in the ranges of 55-64% and 25-48% at the levels of nucleotide and amino acid sequences, respectively. In previous studies, describing multiple RHOs in sphingomonads, substrate preferences of most of the RHOs have not been studied at length. Rather, the degradative genes have been annotated as bph, phn or ahd genes, merely on the basis of the aromatic compounds degraded by the individual species. A closer look at the phylogenetic tree of α-subunits reveals that the clustering depends broadly on substrate specificities [6]. Tree topology indicates that the homologous proteins first branch according to their substrate class preferences and within each branch, more similar sequences group in accordance with substrate sub-classes and ultimately, cluster according to the species tree. It has been observed that each of the homologous α-subunit proteins from sphingomonads clusters together in different clades (Fig. 2). Apart from the homologous α-subunit proteins from sphingomonads (78–100% identity), few homologous α-subunits were also detected from the whole genome sequence of Cycloclasticus sp. P1, which showed up to 64% sequence identity to the α-subunit proteins determined in strain PNB. According to the classification suggested by Chakraborty et al.[6], AhdA1b and AhdA1f of strain PNB and their homologues belong to A-IIIαβ type RHOs where AhdA1f corresponds to well studied PAH dioxygenases in sphingomonads [7,10,19] while AhdA1b correspond to ethylbenzene dioxygenase (72.8% identity) in Rhodococcus jostii RHA1 [20], one of the least explored A-IIIαβ type RHOs. On the other hand, XylX has largely been described as benzoate/toluate dioxygenase belonging to B-IIαβ type RHO [8]. The α-subunit from strain PNB, designated as xylX, also clustered with benzoate/toluate dioxygenases present in various genera and showed maximum identity (50.8%) with the well characterized benzoate dioxygenase from Pseudomonas putida[21]. On the other hand, AhdA1c, AhdA1d, and AhdA1e, all of which belong to C-IVαβ type RHOs, branched into three different subclusters (Fig. 2). AhdA1c and AhdA1d showed 49.75 and 47.25% identity with biochemically characterized o-halobenzoate dioxygenases of Achromobacter xylosoxidans A8 [22] and Burkholderia mallei ATCC 23344 [23] respectively. Similarly, AhdA1e clustered distinctly along with that of the homologous α-subunits from other sphingomonads and shared a common ancestry with o-halobenzoate dioxygenase and salicylate 5-hydroxylase.


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)

Dendogram showing the relatedness of α-subunit of bacterial aromatic RHOs along with the known biochemical reactions catalyzed. Class A, B and C RHOs (following classification scheme as described by Chakraborty et al.[6]) are shown in shades of green, brown and blue, respectively. Each shade within a class represents different reaction chemistry (with respect to oxygenation sites) while the lightest shade in each class represents the α-subunit belonging to sphingomonads. Values at each node indicate level of bootstrap support based on 100 resampled datasets while bootstrap values below 50% are not shown. A Class D carbazole dioxygenase (CarAaI) from Sphingomonas sp. KA1 (GenBank: YP_717981) was used as outgroup. (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

f0010: Dendogram showing the relatedness of α-subunit of bacterial aromatic RHOs along with the known biochemical reactions catalyzed. Class A, B and C RHOs (following classification scheme as described by Chakraborty et al.[6]) are shown in shades of green, brown and blue, respectively. Each shade within a class represents different reaction chemistry (with respect to oxygenation sites) while the lightest shade in each class represents the α-subunit belonging to sphingomonads. Values at each node indicate level of bootstrap support based on 100 resampled datasets while bootstrap values below 50% are not shown. A Class D carbazole dioxygenase (CarAaI) from Sphingomonas sp. KA1 (GenBank: YP_717981) was used as outgroup. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Mentions: Sequence analysis revealed seven sets of putative α- and β-subunit RHO genes, with one of the α-subunits (ahdA1a) disrupted. Each α subunit contains an N-terminal ISP domain, with a conserved Rieske [2Fe–2S] center, a C-terminal catalytic domain having a conserved mononuclear iron-binding site and a conserved aspartate, which is known to facilitate inter-subunit electron transfer between ISP and catalytic domains of α-subunits [14,17,18]. The genes encoding β-subunits were found adjacent to that of the α-subunits in all the sets of oxygenases indicating possible co-evolution of α- and β-subunits and the presence of hetero-multimeric (αnβn)-type of RHOs in strain PNB. Fig. 2 illustrates the phylogenetic relation of the α-subunit protein sequences (AhdA1b, AhdA1c, AhdA1d, AhdA1e, AhdA1f and XylX) in strain PNB and the homologous sequences from other organisms, as mentioned in Table S3. Although the α-subunits in strain PNB share conserved domain regions, their nucleotide sequences and deduced amino acid sequences share limited homology with that of the nonsphingomonad counterparts. Moreover, phylogenetic analysis reveals that the individual α-subunit proteins in sphingomonads are distantly related (Fig. 2). Pairwise sequence alignments among the α-subunit in strain PNB, showed identities in the ranges of 55-64% and 25-48% at the levels of nucleotide and amino acid sequences, respectively. In previous studies, describing multiple RHOs in sphingomonads, substrate preferences of most of the RHOs have not been studied at length. Rather, the degradative genes have been annotated as bph, phn or ahd genes, merely on the basis of the aromatic compounds degraded by the individual species. A closer look at the phylogenetic tree of α-subunits reveals that the clustering depends broadly on substrate specificities [6]. Tree topology indicates that the homologous proteins first branch according to their substrate class preferences and within each branch, more similar sequences group in accordance with substrate sub-classes and ultimately, cluster according to the species tree. It has been observed that each of the homologous α-subunit proteins from sphingomonads clusters together in different clades (Fig. 2). Apart from the homologous α-subunit proteins from sphingomonads (78–100% identity), few homologous α-subunits were also detected from the whole genome sequence of Cycloclasticus sp. P1, which showed up to 64% sequence identity to the α-subunit proteins determined in strain PNB. According to the classification suggested by Chakraborty et al.[6], AhdA1b and AhdA1f of strain PNB and their homologues belong to A-IIIαβ type RHOs where AhdA1f corresponds to well studied PAH dioxygenases in sphingomonads [7,10,19] while AhdA1b correspond to ethylbenzene dioxygenase (72.8% identity) in Rhodococcus jostii RHA1 [20], one of the least explored A-IIIαβ type RHOs. On the other hand, XylX has largely been described as benzoate/toluate dioxygenase belonging to B-IIαβ type RHO [8]. The α-subunit from strain PNB, designated as xylX, also clustered with benzoate/toluate dioxygenases present in various genera and showed maximum identity (50.8%) with the well characterized benzoate dioxygenase from Pseudomonas putida[21]. On the other hand, AhdA1c, AhdA1d, and AhdA1e, all of which belong to C-IVαβ type RHOs, branched into three different subclusters (Fig. 2). AhdA1c and AhdA1d showed 49.75 and 47.25% identity with biochemically characterized o-halobenzoate dioxygenases of Achromobacter xylosoxidans A8 [22] and Burkholderia mallei ATCC 23344 [23] respectively. Similarly, AhdA1e clustered distinctly along with that of the homologous α-subunits from other sphingomonads and shared a common ancestry with o-halobenzoate dioxygenase and salicylate 5-hydroxylase.

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