Abstract
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.
Original language | English (US) |
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Pages (from-to) | 733-740 |
Number of pages | 8 |
Journal | Journal of Biological Inorganic Chemistry |
Volume | 8 |
Issue number | 7 |
DOIs | |
State | Published - Sep 2003 |
All Science Journal Classification (ASJC) codes
- Biochemistry
- Inorganic Chemistry
Keywords
- Cytochrome P450
- Hydroxylase
- Methane monooxygenase
- Non-heme diiron enzymes
- Radical clocks