Profiling Mechanisms of Alkane Hydroxylase Activity In Vivo Using the Diagnostic Substrate Norcarane

Elena A. Rozhkova-Novosad; Jong Chan Chae; Gerben J. Zylstra; Erin M. Bertrand; Marselle Alexander-Ozinskas; Dayi Deng; Luke A. Moe; Jan B. van Beilen; Michael Danahy; John Taylor Groves; Rachel N. Austin

doi:10.1016/j.chembiol.2006.12.007

Profiling Mechanisms of Alkane Hydroxylase Activity In Vivo Using the Diagnostic Substrate Norcarane

Elena A. Rozhkova-Novosad
, Jong Chan Chae
, Gerben J. Zylstra
, Erin M. Bertrand
, Marselle Alexander-Ozinskas
, Dayi Deng
, Luke A. Moe
, Jan B. van Beilen
, Michael Danahy
, John Taylor Groves
, Rachel N. Austin

Research output: Contribution to journal › Article › peer-review

32 Scopus citations

Abstract

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.

Original language	English (US)
Pages (from-to)	165-172
Number of pages	8
Journal	Chemistry and Biology
Volume	14
Issue number	2
DOIs	https://doi.org/10.1016/j.chembiol.2006.12.007
State	Published - Feb 2007

All Science Journal Classification (ASJC) codes

Biochemistry
Molecular Medicine
Molecular Biology
Pharmacology
Drug Discovery
Clinical Biochemistry

Keywords

CHEMBIOL

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10.1016/j.chembiol.2006.12.007

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Rozhkova-Novosad, E. A., Chae, J. C., Zylstra, G. J., Bertrand, E. M., Alexander-Ozinskas, M., Deng, D., Moe, L. A., van Beilen, J. B., Danahy, M., Groves, J. T., & Austin, R. N. (2007). Profiling Mechanisms of Alkane Hydroxylase Activity In Vivo Using the Diagnostic Substrate Norcarane. Chemistry and Biology, 14(2), 165-172. https://doi.org/10.1016/j.chembiol.2006.12.007

@article{d1b0f2497ed64b3fb672d41181f00524,

title = "Profiling Mechanisms of Alkane Hydroxylase Activity In Vivo Using the Diagnostic Substrate Norcarane",

abstract = "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.",

keywords = "CHEMBIOL",

author = "Rozhkova-Novosad, \{Elena A.\} and Chae, \{Jong Chan\} and Zylstra, \{Gerben J.\} and Bertrand, \{Erin M.\} and Marselle Alexander-Ozinskas and Dayi Deng and Moe, \{Luke A.\} and \{van Beilen\}, \{Jan B.\} and Michael Danahy and Groves, \{John Taylor\} and Austin, \{Rachel N.\}",

note = "Funding 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.",

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doi = "10.1016/j.chembiol.2006.12.007",

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T1 - Profiling Mechanisms of Alkane Hydroxylase Activity In Vivo Using the Diagnostic Substrate Norcarane

AU - Rozhkova-Novosad, Elena A.

AU - Chae, Jong Chan

AU - Zylstra, Gerben J.

AU - Bertrand, Erin M.

AU - Alexander-Ozinskas, Marselle

AU - Deng, Dayi

AU - Moe, Luke A.

AU - van Beilen, Jan B.

AU - Danahy, Michael

AU - Groves, John Taylor

AU - Austin, Rachel N.

N1 - Funding 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.

PY - 2007/2

Y1 - 2007/2

N2 - 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.

AB - 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.

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U2 - 10.1016/j.chembiol.2006.12.007

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JF - Chemistry and Biology

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ER -

Profiling Mechanisms of Alkane Hydroxylase Activity In Vivo Using the Diagnostic Substrate Norcarane

Abstract

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