Purification and characterization of a three-component salicylate 1-hydroxylase from Sphingomonas CHY-1
Résumé
In the bacterial degradation of polycyclic aromatic hydrocarbons (PAHs), salicylate hydroxylases catalyze essential reactions at the junction between the so-called upper and lower catabolic pathways. Unlike the salicylate 1-hydroxylase from pseudomonads, which is a well-characterized flavoprotein, the enzyme found in sphingomonads appears to be a three-component Fe-S protein complex, which has not so far been characterized. Here, the salicylate 1-hydroxylase from Sphingomonas CHY-1 has been purified and characterized with respect to its biochemical and catalytic properties. The oxygenase component called PhnII, exhibited an α3β3 heterohexameric structure and contained one Rieske-type [2Fe-2S] cluster and one mononuclear iron per α subunit. In the presence of purified reductase (PhnA4) and ferredoxin (PhnA3) components, PhnII catalyzed the hydroxylation of salicylate to catechol with a maximal specific activity of 0.89 U/mg and showed an apparent Km for salicylate of 1.1 ± 0.2 µM. The hydroxylase exhibited similar activity levels with methylsalicylates and low activity with salicylate analogues bearing additional hydroxyl or electron-withdrawing substituents. PhnII converted anthranilate to 2-aminophenol and exhibited a relatively low affinity for this substrate (Km = 28 ± 6 µM). 1-Hydroxy-2-naphthoate, which is an intermediate in phenanthrene degradation, was not hydroxylated by PhnII, but induced a high rate of uncoupled oxidation of NADH. It also exerted a strong competitive inhibition of salicylate hydroxylation, with a Ki of 0.68 µM. The properties of this three-component hydroxylase are compared with those of analogous bacterial hydroxylases and discussed in the light of our current knowledge of PAH degradation by sphingomonads.
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