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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 alpha  and beta  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 alpha  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 alpha  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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