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Purification and characterization of a three-component salicylate 1-hydroxylase from Sphingomonas sp. strain CHY-1.
| Content Provider | Semantic Scholar |
|---|---|
| Author | Jouanneau, Yves Micoud, Julien Meyer, Christine |
| Copyright Year | 2007 |
| 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. |
| File Format | PDF HTM / HTML |
| DOI | 10.1128/AEM.01519-07 |
| PubMed reference number | 17905882 |
| Journal | Medline |
| Volume Number | 73 |
| Issue Number | 23 |
| Alternate Webpage(s) | http://aem.asm.org/content/73/23/7515.full.pdf |
| Alternate Webpage(s) | https://doi.org/10.1128/AEM.01519-07 |
| Journal | Applied and environmental microbiology |
| Language | English |
| Access Restriction | Open |
| Content Type | Text |
| Resource Type | Article |
