PC 2414 Green Heme

In an attempt to rlucidatc the mrrhnnism of the enzymic hydrosylation of aromatic compounds, we have isolated and partislly purified an aromatic hydrosglase from a soil bacterium. This enzyme, which will be referred to as “salicylate hydroxylase,” is soluble and relatively stable, in contrast to most hitherto known aromatic hydrosylascs, and ratalyzes a stoichiometric conversion of salicylate ‘co raterhol in the presence of reduced diphosphopyridine nuclcotidc and osygcn. iZfter acid ammonium sulfate treatment of the enzyme, its catalytic activity was shown to require the addition of a fartor present in boiled juice of rat liver that has now been identified as flavin adenine dinurleotide. A brief description of the experimental evidence and the implication of thcsc findings are reported here. The cells of a gram-positive coccus, which was isolated from soil b,v the enrichment technique (l), were grown for approximately 30 hours at 30”, with constant shaking, in a medium containing 0.1 y0 ?jH&TOs, 0.1% sodium salicylate, 0.1 5yG KzHP04, O.O5c/, KH2P04, 0.02%, Mg80.1~7Hz0, and 0.3 mg per liter of riboflavin. Cell-free r&acts were prepared by subjecting the (~11 suspension to the action of a Kubota lo-kc sonic oscillator for 15 minutes, followed by crntrifugation at 104,000 X g for 60 minutes. To 100 ml of the sul)ernatant solution containing usually approximately 10 mg of protein per ml were added 100 ml of a saturated ammonium sulfate solution, and the pH of the mixture was adjusted to 2.7 by the addition of 0.5 N HCl. The precipitatr that formrd after the misturc had been left overnight in a refrigerator \vas collcctcd by ccntrifugation, washed once with apl,rosimately 100 ml of a 6Oo/;, saturated ammonium sulfate solution (pH 2.7), and dissolved in 30 ml of 0.5 M phosphate buffer, pH 8.0. The enzyme solution thus obtained was dialyzed against a large amount of 33 mM phosphate buffer, pH 7.0, to remove ammonium ion, which is inhibitory to the enzyme activity. As shown in Table I, 1 mole of cattchol was produced from 1 mole of salicylate with the consumption of approximately 1 mole of DPKH. TPNH showed apyroximatcly 80% the activity of DPNH under the conditions tcsttd. In the absence of FAD, practically no reaction took place. F1\2N,’ riboflavin, tetrahydrofolic acid,% or ferrous sulfate did not replace FL4D. Reducing agents such as GSH, ascorbic acid, tctrahydrofolic acid, and ferrous sulfate were ineffective in place of DPNH. 9 parallel experiment carried out in a Warburg apparatus indicated a stoichiometric consumption of oxygen and formation of Con. 2,3-Dihydrosybenzoate was also metabolized by this enzyme. Phenol, benzoate, and anthranilatr, however, were completely inert as substrate.