Reconstitution of active mycobacterial binuclear iron monooxygenase complex in escherichia coli

Toshiki Furuya, Mika Hayashi, Kuniki Kino

    Research output: Contribution to journalArticle

    5 Citations (Scopus)

    Abstract

    Bacterial binuclear iron monooxygenases play numerous physiological roles in oxidative metabolism. Monooxygenases of this type found in actinomycetes also catalyze various useful reactions and have attracted much attention as oxidation biocatalysts. However, difficulties in expressing these multicomponent monooxygenases in heterologous hosts, particularly in Escherichia coli, have hampered the development of engineered oxidation biocatalysts. Here, we describe a strategy to functionally express the mycobacterial binuclear iron monooxygenase MimABCD in Escherichia coli. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of the mimABCD gene expression in E. coli revealed that the oxygenase components MimA and MimC were insoluble. Furthermore, although the reductase MimB was expressed at a low level in the soluble fraction of E. coli cells, a band corresponding to the coupling protein MimD was not evident. This situation rendered the transformed E. coli cells inactive. We found that the following factors are important for functional expression of MimABCD in E. coli: coexpression of the specific chaperonin MimG, which caused MimA and MimC to be soluble in E. coli cells, and the optimization of the mimD nucleotide sequence, which led to efficient expression of this gene product. These two remedies enabled this multicomponent monooxygenase to be actively expressed in E. coli. The strategy described here should be generally applicable to the E. coli expression of other actinomycetous binuclear iron monooxygenases and related enzymes and will accelerate the development of engineered oxidation biocatalysts for industrial processes.

    Original languageEnglish
    Pages (from-to)6033-6039
    Number of pages7
    JournalApplied and Environmental Microbiology
    Volume79
    Issue number19
    DOIs
    Publication statusPublished - 2013

    Fingerprint

    Mixed Function Oxygenases
    Iron
    iron
    Escherichia coli
    oxidation
    gene expression
    Acinetobacter
    gel
    metabolism
    sodium
    Enzymes
    enzyme
    sulfate
    protein
    gene
    Chaperonins
    chaperonins
    Gene Expression
    Oxygenases
    Actinobacteria

    ASJC Scopus subject areas

    • Applied Microbiology and Biotechnology
    • Food Science
    • Biotechnology
    • Ecology

    Cite this

    Reconstitution of active mycobacterial binuclear iron monooxygenase complex in escherichia coli. / Furuya, Toshiki; Hayashi, Mika; Kino, Kuniki.

    In: Applied and Environmental Microbiology, Vol. 79, No. 19, 2013, p. 6033-6039.

    Research output: Contribution to journalArticle

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