Mechanistically informative chemical probes are used to characterize the activity of functional alkane hydroxylases in whole cells. Norcarane is a substrate used to reveal the lifetime of radical intermediates formed during alkane oxidation. Results from oxidations of this probe with organisms that contain the two most prevalent medium-chain-length alkane-oxidizing metalloenzymes, alkane ω-monooxygenase (AlkB) and cytochrome P450 (CYP), are reported. The results-radical lifetimes of 1-7 ns for AlkB and less than 100 ps for CYP-indicate that these two classes of enzymes are mechanistically distinguishable and that whole-cell mechanistic assays can identify the active hydroxylase. The oxidation of norcarane by several recently isolated strains (Hydrocarboniphaga effusa AP103, rJ4, and rJ5, whose alkane-oxidizing enzymes have not yet been identified) is also reported. Radical lifetimes of 1-3 ns are observed, consistent with these organisms containing an AlkB-like enzyme and inconsistent with their employing a CYP-like enzyme for growth on hydrocarbons.
|Number of pages||8|
|Journal||Chemistry and Biology|
|State||Published - Feb 2007|
Bibliographical noteFunding Information:
Financial support from the National Science Foundation through the Environmental Molecular Science Institute CEBIC (Center for Environmental Bioinorganic Chemistry at Princeton University) CHE-0221978 (G.J.Z., R.N.A., and J.T.G.), CHE-0316301 (J.T.G.), MCB-0078465, and CHE-0116233 (R.N.A.); the Camille and Henry Dreyfus Foundation (R.N.A.); and the National Institutes of Health GM-32698 (J.T.G.) and GM072506 (R.N.A.) is gratefully acknowledged. We also thank Dr. John Eng and Dr. Dorothy Little for expert technical assistance with mass spectrometry and Charlotte Lehmann for technical assistance with cell culturing and experiments.
ASJC Scopus subject areas
- Molecular Medicine
- Molecular Biology
- Drug Discovery
- Clinical Biochemistry