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

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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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Notes

  1. This paper reported 100% rearrangement of methyl(phenyl)cyclopropane with partially purified AlkB. While these results are consistent with a radical mechanism, a cationic pathway would have also produced the same product distribution

Abbreviations

AlkB:

alkane monooxygenase

CYP:

cytochrome P450

IPTG:

isopropyl-β-d-thiogalactopyranoside

sMMO:

soluble methane monooxygenase

XylM:

xylene monooxygenase

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Acknowledgements

We are grateful to the National Science Foundation for funding of this research through the Environmental Molecular Science Institute, CEBIC (Center for Environmental Bioinorganic Chemistry) NSF9810248 (J.T.G., G.J.Z., and R.N.A.), CHE9814301 (J.T.G.), and CHE9950314 (R.N.A.). E.K. was supported through a KOSEF grant to the Center for Proteinaceous Materials at Chosun National University. K.B. was supported by a summer undergraduate research fellowship through CEBIC and received travel funds to support her presentation of this work at ICBIC X from the Hughes Foundation. We thank Dr. Zhengbo Hu for supplying us with authentic samples of endo- and exo-3-norcaranol and 3-norcaranone.

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Author notes

  1. Kate Buzzi

    Present address: Medical School, Drexel University, Philadelphia, PA, USA

Authors and Affiliations

  1. Department of Chemistry, Bates College, 5 Andrews Road, Lewiston, ME 04240, USA

    Rachel N. Austin & Kate Buzzi

  2. Department of Biology, Yonsei University, Seoul, South Korea

    Eungbin Kim

  3. Biotechnology Center for Agriculture and the Environment, Rutgers University, New Brunswick, NJ 08901, USA

    Gerben J. Zylstra

  4. Department of Chemistry, Princeton University, Princeton, NJ 08544, USA

    John T. Groves

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  1. Rachel N. Austin
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  2. Kate Buzzi
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Correspondence to Rachel N. Austin or John T. Groves.

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Austin, R.N., Buzzi, K., Kim, E. et al. Xylene monooxygenase, a membrane-spanning non-heme diiron enzyme that hydroxylates hydrocarbons via a substrate radical intermediate. J Biol Inorg Chem 8, 733–740 (2003). https://doi.org/10.1007/s00775-003-0466-3

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