Bacterial metabolism of naphthalene: Construction and use of recombinant bacteria to study ring cleavage of 1,2-dihydroxynaphthalene and subsequent reactions

Technical Report · Wed Jan 01 00:00:00 EST 1992

OSTI ID:6501575

Eaton, R W; Chapman, P J

The reactions involved in the bacterial metabolism of naphthalene to salicylate have been reinvestigated by using recombinant bacteria carrying genes cloned from plasmid NAH7. When intact cells of Pseudomonas aeruginosa PAO1 carrying DNA fragments encoding the first three enzymes of the pathway were incubated with naphthalene, they formed products of the dioxygenase-catalyzed ring cleavage of 1,2-dihydroxynaphthalene. These products were separated by chromatography on Sephadex G-25 and were identified by (1)H and (13)C nuclear magnetic resonance spectroscopy and gas chromatography-mass spectrometry as 2-hydroxychromene-2-carboxylate (HCCA) and trans-o-hydroxybenzylidenepyruvate (tHBPA). HCCA was detected as the first reaction product in these incubation mixtures by its characteristic UV spectrum, which slowly changed to a spectrum indicative of an equilibrium mixture of HCCA and tHBPA. Isomerization of either purified product occurred slowly and spontaneously to give an equilibrium mixture of essentially the same composition. tHBPA is also formed from HCCA by the action of an isomerase enzyme encoded by plasmid NAH7. The gene encoding this enzyme, nahD, was cloned on a 1.95-kb KpnI-Bg/II fragment. Extracts of Escherichia coli JM109 carrying this fragment catalyzed the rapid equilibration of HCCA and tHBPA. Metabolism of tHBPA to salicylaldehyde by hydration and aldol cleavage is catalyzed by a single enzyme encoded by a 1-kb MluI-StuI restriction fragment. A mechanism for the hydratase-aldolase-catalyzed reaction is proposed. (Copyright (c) 1992 American Society for Microbiology.)

• Research Organization: Environmental Protection Agency, Gulf Breeze, FL (United States). Environmental Research Lab.
• OSTI ID: 6501575
• Report Number(s): PB-93-168938/XAB; EPA-600/J-93/059; CNN: NSF-USE90-50802
• Resource Relation: Other Information: Pub. in Jnl. of Bacteriology, Vol. 174, No. 23, 7542-7554(Dec 1992)
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
37 INORGANIC
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PHYSICAL AND ANALYTICAL CHEMISTRY
GENES
CLONING
METABOLITES
GAS CHROMATOGRAPHY
NMR SPECTRA
NAPHTHALENE
BIODEGRADATION
ENZYMES
ESCHERICHIA COLI
PLASMIDS
PSEUDOMONAS
RECOMBINANT DNA
AROMATICS
BACTERIA
CELL CONSTITUENTS
CHEMICAL REACTIONS
CHROMATOGRAPHY
CONDENSED AROMATICS
DECOMPOSITION
DNA
HYDROCARBONS
MICROORGANISMS
NUCLEIC ACIDS
ORGANIC COMPOUNDS
PROTEINS
SEPARATION PROCESSES
SPECTRA
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550400 - Genetics
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