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

28 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

Keywords

  • CHEMBIO
  • MICROBIO
  • PROTEINS

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Medicine
  • Molecular Biology
  • Pharmacology
  • Drug Discovery
  • Clinical Biochemistry

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