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 omega-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.
authors.affiliation: Department of Chemistry, Princeton University, Princeton, NJ 08544, USA.
authors.forename: Elena A
authors.initials: EA
authors.lastname: Rozhkova-Novosad
authors.forename: Jong-Chan
authors.initials: JC
authors.lastname: Chae
authors.forename: Gerben J
authors.initials: GJ
authors.lastname: Zylstra
authors.forename: Erin M
authors.initials: EM
authors.lastname: Bertrand
authors.forename: Marselle
authors.initials: M
authors.lastname: Alexander-Ozinskas
authors.forename: Dayi
authors.initials: D
authors.lastname: Deng
authors.forename: Luke A
authors.initials: LA
authors.lastname: Moe
authors.forename: Jan B
authors.initials: JB
authors.lastname: van Beilen
authors.forename: Michael
authors.initials: M
authors.lastname: Danahy
authors.forename: John T
authors.initials: JT
authors.lastname: Groves
authors.forename: Rachel N
authors.initials: RN
authors.lastname: Austin
mesh_terms: D016956:Burkholderia cepacia / Q000201:enzymology* / Q000378:metabolism; D042926:Cytochrome P-450 CYP4A / Q000378:metabolism*; D016958:Pseudomonas putida / Q000201:enzymology* / Q000378:metabolism; D012240:Rhodococcus / Q000201:enzymology* / Q000378:metabolism; D013729:Terpenes / Q000378:metabolism*
chemical_list: D013729:Terpenes; D042926:Cytochrome P-450 CYP4A; C460459:norcarane