Xylene monooxygenase, a membrane-spanning non-heme diiron enzyme that hydroxylates hydrocarbons via a substrate radical intermediate

Rachel N. Austin, Kate Buzzi, Eungbin KIm, Gerben J. Zylstra, John T. Groves

Research output: Contribution to journalArticle

20 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)733-740
Number of pages8
JournalJournal of Biological Inorganic Chemistry
Volume8
Issue number7
Publication statusPublished - 2003 Sep 1

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Hydrocarbons
Pseudomonas putida
Clone Cells
Mixed Function Oxygenases
Membranes
Assays
Substrates
Enzymes
Carbon
Genes
Sphingomonas
Isopropyl Thiogalactoside
Heptanes
Carbon Cycle
Oxygenases
Escherichia coli
Genetic Recombination
Clocks
Hydrogen
Screening

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Inorganic Chemistry

Cite this

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abstract = "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.",
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Xylene monooxygenase, a membrane-spanning non-heme diiron enzyme that hydroxylates hydrocarbons via a substrate radical intermediate. / Austin, Rachel N.; Buzzi, Kate; KIm, Eungbin; Zylstra, Gerben J.; Groves, John T.

In: Journal of Biological Inorganic Chemistry, Vol. 8, No. 7, 01.09.2003, p. 733-740.

Research output: Contribution to journalArticle

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