Metabolism of Aromatic Compounds in Bacteria PURIFICATIOK AND PROPERTIES OF THE CATECHOL-FORMING

The conversion of 3,5-cyclohexadiene-1 J-diol-l-carboxylic acid (DHB) to catechol has been shown to be catalyzed by a single protein from the bacterium Alcaligenes eutrophus. The enzyme was purified by ultracentrifugation, ammonium sulfate fractionation, DEAE-cellulose chromatography and Sephadex G-ZOO gel filtration. Its molecular weight is approximately 94,600 (by sedimentation equilibrium) and the molecular weight of its subunits is approximately 24,000 (by sodium dodecyl sulfate gel electrophoresis). The proposed mechanism of the reaction is dehydrogenation of DHB to an unstable /3-ketoacid which decarboxylates to form catechol. benzene-methanol-acetic acid. (RF for benzoic acid, 0.67.) [C~rbozyl-~~C]DHB was prepared biologically by modifying, as follows, the procedure for preparation of unlabeled DHB (5). Five microcuries of purified [carboxy&14C]benzoic acid (56 PCi per pmole) was incubated with an aerated l-ml culture of strain B9 (IO9 cells per ml) for 15 min at 30”. The culture supernatant fluid was extracted with ethyl acetate, and the organic phase was concentrated under a stream of nitrogen. [l%]DHB was purified from this by thin layer chromatography in the solvent system described above. (RF for DHB, 0.23.) Cis-3,5-cyclohexadiene-1,2-dial was a gift from Dr. D. T. Gibson. NAD+, NADH, NADPf, ATP, yeast alcohol dehydrogenase, and DEAEcellulose were from Sigma. Ammonium sulfate was ultrapure grade from Mann. Hyamine was from Nuclear Chicago. Serum caps and plastic center wells for measurement of 14C02 release were from Kontes, Vineland, New Jersey.