title: Purification and characterization of a three-component salicylate 1-hydroxylase from Sphingomonas sp. strain CHY-1.
abstract: 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 so far has not been characterized. Here, the salicylate 1-hydroxylase from Sphingomonas sp. strain CHY-1 was purified, and its biochemical and catalytic properties were characterized. The oxygenase component, designated PhnII, exhibited an alpha3beta3 heterohexameric structure and contained one Rieske-type [2Fe-2S] cluster and one mononuclear iron per alpha 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 microM. 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 microM). 1-Hydroxy-2-naphthoate, which is an intermediate in phenanthrene degradation, was not hydroxylated by PhnII, but it induced a high rate of uncoupled oxidation of NADH. It also exerted strong competitive inhibition of salicylate hydroxylation, with a Ki of 0.68 microM. The properties of this three-component hydroxylase are compared with those of analogous bacterial hydroxylases and are discussed in light of our current knowledge of PAH degradation by sphingomonads.
authors: LCBM/iRTSV, CEA-Grenoble, F-38054 Grenoble Cedex 9, France. yves.jouanneau@cea.fr
authors: Yves
authors: Y
authors: Jouanneau
authors: Julien
authors: J
authors: Micoud
authors: Christine
authors: C
authors: Meyer
mesh_terms: D002384:Catalysis / Q000187:drug effects; D002853:Chromatography, Liquid; D004492:Edetic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D004795:Enzyme Stability / Q000187:drug effects; D007477:Ions / Q000494:pharmacology; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D011522:Protons; D012459:Salicylates / Q000378:metabolism; D020578:Sphingomonas / Q000201:enzymology*; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D007477:Ions; D011522:Protons; D012459:Salicylates; D004492:Edetic Acid; D006899:Mixed Function Oxygenases; C022619:salicylate 1-monooxygenase; C033069:methyl salicylate