The non-heme diiron enzyme xylene monooxygenase (XylM) has been shown to hydroxylate hydrocarbons via a hydrogen abstraction-carbon radical recombination mechanism (oxygen rebound). Using the radical clock bicyclo[4.1.0]heptane (norcarane) in a whole-cell assay, and observing the ratio of rearranged 3-(hydroxymethyl)cyclohexene and unrearranged 2-norcaranol products, the lifetime of the substrate radical was determined to be approximately 0.2 ns. The wild-type organism Pseudomonas putida mt-2 and two separate Escherichia coli clones expressing xylMA genes gave similar results. One clone produced the Pseudomonas putida mt-2 XylMA hydroxylase and the other produced Sphingomonas yanoikuyae B1 XylMA hydroxylase. Clones were constructed by inserting genes for xylene monooxygenase and xylene monooxygenase reductase downstream from an IPTG-inducible T7 promoter. Mechanistic investigations using whole-cell assays will facilitate more rapid screening of structure-function relationships and the identification of novel oxygenases. This approach should enable the construction of a picture of the key metalloenzymes and the mechanisms they use in selected parts of the global carbon cycle without requiring the isolation of every protein involved.
|Number of pages||8|
|Journal||Journal of Biological Inorganic Chemistry|
|Publication status||Published - 2003 Sep 1|
All Science Journal Classification (ASJC) codes
- Inorganic Chemistry