Xylene monooxygenase, a membrane-spanning non-heme diiron enzyme that hydroxylates hydrocarbons via a substrate radical intermediate.
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.
Austin
Rachel N
RN
Department of Chemistry, Bates College, 5 Andrews Road, Lewiston, ME 04240, USA. raustin@bates.edu
Buzzi
Kate
K
Kim
Eungbin
E
Zylstra
Gerben J
GJ
Groves
John T
JT
D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D002384:Catalysis; D004926:Escherichia coli / Q000201:enzymology; D005609:Free Radicals; D006838:Hydrocarbons / Q000737:chemistry / Q000378:metabolism*; D006900:Hydroxylation; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D008667:Metalloproteins / Q000737:chemistry / Q000378:metabolism; D010105:Oxygenases / Q000737:chemistry / Q000378:metabolism*; D016958:Pseudomonas putida / Q000201:enzymology; D013729:Terpenes
D001426:Bacterial Proteins; D005609:Free Radicals; D006838:Hydrocarbons; D008565:Membrane Proteins; D008667:Metalloproteins; D013729:Terpenes; D010105:Oxygenases; C067624:xylene monooxygenase; C460459:norcarane