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.
Bibliographical noteFunding Information:
Acknowledgements We are grateful to the National Science Foundation for funding of this research through the Environmental Molecular Science Institute, CEBIC (Center for Environmental Bioinorganic Chemistry) NSF9810248 (J.T.G., G.J.Z., and R.N.A.), CHE9814301 (J.T.G.), and CHE9950314 (R.N.A.). E.K. was supported through a KOSEF grant to the Center for Proteinaceous Materials at Chosun National University. K.B. was supported by a summer undergraduate research fellowship through CEBIC and received travel funds to support her presentation of this work at ICBIC X from the Hughes Foundation. We thank Dr. Zhengbo Hu for supplying us with authentic samples of endo-and exo-3-norcaranol and 3-norcaranone.
All Science Journal Classification (ASJC) codes
- Inorganic Chemistry