Characterization of Orphan Monooxygenases by Rapid Substrate Screening Using FT-ICR Mass Spectrometry

Toshiki Furuya, Tatsunari Nishi, Daisuke Shibata, Hideyuki Suzuki, Daisaku Ohta, Kuniki Kino

    Research output: Contribution to journalArticle

    27 Citations (Scopus)

    Abstract

    Characterization of orphan enzymes, for which the catalytic functions and actual substrates are still not elucidated, is a significant challenge in the postgenomic era. Here, we describe a general strategy for exploring the catalytic potentials of orphan monooxygenases based on direct infusion analysis by Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR/MS). Eight cytochromes P450 from Bacillus subtilis were recombinantly expressed in Escherichia coli and subjected to a reconstitution system containing appropriate electron transfer components and many potential substrates. The reaction mixtures were directly analyzed using FT-ICR/MS, and substrates of the putative enzymes were readily identified from the mass spectral data. This allowed identification of previously unreported CYP109B1 substrates and the functional assignment of two putative cytochromes P450, CYP107J1 and CYP134A1. The FT-ICR/MS-based approach can be easily applied to large-scale screening with the aid of the extremely high mass resolution and accuracy.

    Original languageEnglish
    Pages (from-to)563-572
    Number of pages10
    JournalChemistry and Biology
    Volume15
    Issue number6
    DOIs
    Publication statusPublished - 2008 Jun 23

    Fingerprint

    Cyclotrons
    Fourier Analysis
    Mixed Function Oxygenases
    Cyclotron resonance
    Mass spectrometry
    Mass Spectrometry
    Screening
    Ions
    Cytochrome P-450 Enzyme System
    Fourier transforms
    Substrates
    Enzymes
    Bacillus subtilis
    Bacilli
    Electrons
    Escherichia coli

    Keywords

    • CHEMBIO
    • MICROBIO
    • PROTEINS

    ASJC Scopus subject areas

    • Organic Chemistry

    Cite this

    Characterization of Orphan Monooxygenases by Rapid Substrate Screening Using FT-ICR Mass Spectrometry. / Furuya, Toshiki; Nishi, Tatsunari; Shibata, Daisuke; Suzuki, Hideyuki; Ohta, Daisaku; Kino, Kuniki.

    In: Chemistry and Biology, Vol. 15, No. 6, 23.06.2008, p. 563-572.

    Research output: Contribution to journalArticle

    Furuya, Toshiki ; Nishi, Tatsunari ; Shibata, Daisuke ; Suzuki, Hideyuki ; Ohta, Daisaku ; Kino, Kuniki. / Characterization of Orphan Monooxygenases by Rapid Substrate Screening Using FT-ICR Mass Spectrometry. In: Chemistry and Biology. 2008 ; Vol. 15, No. 6. pp. 563-572.
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