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Journal of Bacteriology, May 1999, p. 2675-2682, Vol. 181, No. 9
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
A Novel Aromatic-Ring-Hydroxylating Dioxygenase
from the Diterpenoid-Degrading Bacterium Pseudomonas
abietaniphila BKME-9
Vincent J. J.
Martin and
William W.
Mohn*
Department of Microbiology and Immunology and
Pulp and Paper Center, University of British Columbia, Vancouver,
British Columbia V6T 1Z3, Canada
Received 27 October 1998/Accepted 16 February 1999
Pseudomonas abietaniphila BKME-9 is able to degrade
dehydroabietic acid (DhA) via ring hydroxylation by a novel
dioxygenase. The ditA1, ditA2, and
ditA3 genes, which encode the
and
subunits of the
oxygenase and the ferredoxin of the diterpenoid dioxygenase, respectively, were isolated and sequenced. The ferredoxin gene is 9.2 kb upstream of the oxygenase genes and 872 bp upstream of a putative
meta ring cleavage dioxygenase gene, ditC. A
Tn5 insertion in the
subunit gene, ditA1,
resulted in the accumulation by the mutant strain BKME-941 of the
pathway intermediate, 7-oxoDhA. Disruption of the ferredoxin gene,
ditA3, in wild-type BKME-9 by mutant-allele exchange
resulted in a strain (BKME-91) with a phenotype identical to that of
the mutant strain BKME-941. Sequence analysis of the putative
ferredoxin indicated that it is likely to be a [4Fe-4S]- or
[3Fe-4S]-type ferredoxin and not a [2Fe-2S]-type ferredoxin, as
found in all previously described ring-hydroxylating dioxygenases.
Expression in Escherichia coli of ditA1A2A3,
encoding the diterpenoid dioxygenase without its putative
reductase component, resulted in a functional enzyme. The diterpenoid
dioxygenase attacks 7-oxoDhA, and not DhA, at C-11 and C-12, producing
7-oxo-11,12-dihydroxy-8,13-abietadien acid, which was identified by
1H nuclear magnetic resonance, UV-visible light, and
high-resolution mass spectrometry. The organization of the genes
encoding the various components of the diterpenoid dioxygenase, the
phylogenetic distinctiveness of both the
subunit and the
ferredoxin component, and the unusual Fe-S cluster of the ferredoxin
all suggest that this enzyme belongs to a new class of aromatic
ring-hydroxylating dioxygenases.
*
Corresponding author. Mailing address: Department of
Microbiology and Immunology, University of British Columbia, 300-6174 University Blvd., Vancouver, B.C., Canada V6T 1Z3. Phone: (604) 822-4285. Fax: (604) 822-6041. E-mail:
wmohn{at}interchange.ubc.ca.
Journal of Bacteriology, May 1999, p. 2675-2682, Vol. 181, No. 9
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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