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Main Title Bacterial Metabolism of Naphthalene: Construction and Use of Recombinant Bacteria to Study Ring Cleavage of 1,2-Dihydroxynaphthalene and Subsequent Reactions.
Author Eaton, R. W. ; Chapman, P. J. ;
CORP Author Environmental Research Lab., Gulf Breeze, FL.;National Science Foundation, Washington, DC.
Publisher c1992
Year Published 1992
Report Number EPA/600/J-93/059 ;CONTRIB-795; NSF-USE90-50802;
Stock Number PB93-168938
Additional Subjects Pseudomonas aeruginosa ; Escherichia coli ; Metabolism ; Naphthalene ; Bacterial genes ; Plasmids ; Bacterial DNA ; Genetic recombination ; Liquid chromatography ; Enzymes ; Molecular cloning ; Biotransformation ; Mass fragmentography ; Reprints ; Dihydroxynaphthalenes
Library Call Number Additional Info Location Last
NTIS  PB93-168938 Some EPA libraries have a fiche copy filed under the call number shown. 07/26/2022
Collation 15p
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.)