Sequence analysis has confirmed that the genes coding for the enzymes involved
in DF degradation are dispersed vastly on chromosome. Armengaud et al. (1999)
have presented the genetic organization of the enzymes with several loci. The α and
β subunits of dibenzofuran 4,4a-dioxygenase, DbfB and H1 hydrolase are mapped to
dxnA1A2, dbfB and dxnB gene from Locus A. Locus B and C are associated with
fdx1 and redA2 genes encoding, respectively the ferredoxin and reductase of the
RW1 dibenzofuran dioxygenase. Cosmidic library of RW1 has been hybridized with
a probe (obtained using two primers) to identify the Locus D. These primers have
been designed based on DxnA1 N-terminal end of conserved motives in Class II B
dioxygenases. These primers were designed to amplify dxnA1A2; however, they
amplified a short fragment carrying an ORF that encode a polypeptide. Thus
demonstrating the identity to another ring-hydroxylating dioxygenase (α-subunit).
Later on, analysis of edo2 gene sequence harboured on Locus E, identified additional
ORFs suggesting ring-hydroxylating dioxygenases (α and β subunits) share homology with ORFs G1, G5, G6. It was found that the Locus E harbour several degradative genes that also include monooxygenase (ORF G4). The ORF G4 polypeptide
sequence was found to have similarity with several flavin-containing
monooxygenases when isolated from different sources. The ORF G2 (edo2) and
ORF G4 have similarities to Rieske-type (2Fe-2S) ferredoxins (Class IIB and III
ring-hydroxylating dioxygenases). G4 is located to downstream of ORF encoding
G7 and it shows homology to HOPDA hydrolases.
This demonstrates that the genes dxnA1A2 and dbfB, dxnB and dxnC are located
on Locus A. dxnB is located downstream of dxnC(first ORFs) in a compact operon
(Armengaud et al. 1999). Of the ten ORFs first three are dxnC, ORF2 and fdx3. The
ORF of dxnC encodes a protein that has very low homology to bacterial siderophore
receptors, ORF2 encodes a polypeptide that represents insignificant homology to
known proteins and fdx3 encodes a putative ferredoxin. In vitro assays have
demonstrated that RedA2 reduces the Fdx3. This has been further confirmed by in
vivo assays where co-expression of both fdx3 and redA2 with dxnA1A2 has confirmed that Fdx3 can serve as an electron donor for the dioxin dioxygenase. This
ferredoxin and Fdx1 have similar midpoint redox potential. The gene dxnD downstream of fdx3 gene encodes a phenol monooxygenase-like protein (69- kDa) that
has an activity for the turnover of 4-hydroxysalicylate. The gene dxnD is followed by
dxnE that encodes a 37-kDa protein. This protein shares a similarity in sequence and
activity to maleylacetate reductases. Following dxnE is dxnF that encodes an
intradiol dioxygenase (33-kDa) that cleaves hydroxyquinol to produce
maleylacetate.
Further to dxnF is dxnGH that encode a heteromeric protein is a 3-oxoadipate
succinyl-CoA (coenzyme A) transferase. Following dxnGH is dxnI that encode a
protein that exhibits remarkable homology to thiolases (acetyl-CoA
acetyltransferases) (Armengaud et al. 1999). Finally the last ORF encodes a putative
transposase. Combining together, the five ORFs- dxnD, dxnF, dxnE, dxnGH and
dxnI form a 4-hydroxysalicylate/hydroxyquinol degradative pathway.
Thus Sphingomonas sp. RW1 is genetically diverse with four different α-subunits
of ring-hydroxylating dioxygenase, two α-subunits, two reductases, three
14 Metabolism of Dioxins and Dioxins-Like Compound, Its Regulation and. . .
303
in DF degradation are dispersed vastly on chromosome. Armengaud et al. (1999)
have presented the genetic organization of the enzymes with several loci. The α and
β subunits of dibenzofuran 4,4a-dioxygenase, DbfB and H1 hydrolase are mapped to
dxnA1A2, dbfB and dxnB gene from Locus A. Locus B and C are associated with
fdx1 and redA2 genes encoding, respectively the ferredoxin and reductase of the
RW1 dibenzofuran dioxygenase. Cosmidic library of RW1 has been hybridized with
a probe (obtained using two primers) to identify the Locus D. These primers have
been designed based on DxnA1 N-terminal end of conserved motives in Class II B
dioxygenases. These primers were designed to amplify dxnA1A2; however, they
amplified a short fragment carrying an ORF that encode a polypeptide. Thus
demonstrating the identity to another ring-hydroxylating dioxygenase (α-subunit).
Later on, analysis of edo2 gene sequence harboured on Locus E, identified additional
ORFs suggesting ring-hydroxylating dioxygenases (α and β subunits) share homology with ORFs G1, G5, G6. It was found that the Locus E harbour several degradative genes that also include monooxygenase (ORF G4). The ORF G4 polypeptide
sequence was found to have similarity with several flavin-containing
monooxygenases when isolated from different sources. The ORF G2 (edo2) and
ORF G4 have similarities to Rieske-type (2Fe-2S) ferredoxins (Class IIB and III
ring-hydroxylating dioxygenases). G4 is located to downstream of ORF encoding
G7 and it shows homology to HOPDA hydrolases.
This demonstrates that the genes dxnA1A2 and dbfB, dxnB and dxnC are located
on Locus A. dxnB is located downstream of dxnC(first ORFs) in a compact operon
(Armengaud et al. 1999). Of the ten ORFs first three are dxnC, ORF2 and fdx3. The
ORF of dxnC encodes a protein that has very low homology to bacterial siderophore
receptors, ORF2 encodes a polypeptide that represents insignificant homology to
known proteins and fdx3 encodes a putative ferredoxin. In vitro assays have
demonstrated that RedA2 reduces the Fdx3. This has been further confirmed by in
vivo assays where co-expression of both fdx3 and redA2 with dxnA1A2 has confirmed that Fdx3 can serve as an electron donor for the dioxin dioxygenase. This
ferredoxin and Fdx1 have similar midpoint redox potential. The gene dxnD downstream of fdx3 gene encodes a phenol monooxygenase-like protein (69- kDa) that
has an activity for the turnover of 4-hydroxysalicylate. The gene dxnD is followed by
dxnE that encodes a 37-kDa protein. This protein shares a similarity in sequence and
activity to maleylacetate reductases. Following dxnE is dxnF that encodes an
intradiol dioxygenase (33-kDa) that cleaves hydroxyquinol to produce
maleylacetate.
Further to dxnF is dxnGH that encode a heteromeric protein is a 3-oxoadipate
succinyl-CoA (coenzyme A) transferase. Following dxnGH is dxnI that encode a
protein that exhibits remarkable homology to thiolases (acetyl-CoA
acetyltransferases) (Armengaud et al. 1999). Finally the last ORF encodes a putative
transposase. Combining together, the five ORFs- dxnD, dxnF, dxnE, dxnGH and
dxnI form a 4-hydroxysalicylate/hydroxyquinol degradative pathway.
Thus Sphingomonas sp. RW1 is genetically diverse with four different α-subunits
of ring-hydroxylating dioxygenase, two α-subunits, two reductases, three
14 Metabolism of Dioxins and Dioxins-Like Compound, Its Regulation and. . .
303