Characterization of orphan monooxygenases by rapid substrate screening using FT-ICR mass spectrometry.
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.
Department of Applied Chemistry, Faculty of Science and Engineering, Waseda University, Tokyo, Japan.
Toshiki
T
Furuya
Tatsunari
T
Nishi
Daisuke
D
Shibata
Hideyuki
H
Suzuki
Daisaku
D
Ohta
Kuniki
K
Kino
D001412:Bacillus subtilis / Q000201:enzymology; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D003580:Cytochromes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics; D005583:Fourier Analysis; D013058:Mass Spectrometry / Q000379:methods*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
D003580:Cytochromes; D017931:DNA Primers; D011994:Recombinant Proteins