| RRC ID |
5730
|
| 著者 |
Furuya T, Nishi T, Shibata D, Suzuki H, Ohta D, Kino K.
|
| タイトル |
Characterization of orphan monooxygenases by rapid substrate screening using FT-ICR mass spectrometry.
|
| ジャーナル |
Chem Biol
|
| 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.
|
| 巻・号 |
15(6)
|
| ページ |
563-72
|
| 公開日 |
2008-6-1
|
| DOI |
10.1016/j.chembiol.2008.05.013
|
| PII |
S1074-5521(08)00205-6
|
| PMID |
18559267
|
| MeSH |
Bacillus subtilis / enzymology
Base Sequence
Chromatography, High Pressure Liquid
Cloning, Molecular
Cytochromes / chemistry
Cytochromes / genetics
Cytochromes / metabolism*
DNA Primers
Escherichia coli / genetics
Fourier Analysis
Mass Spectrometry / methods*
Recombinant Proteins / chemistry
Recombinant Proteins / genetics
Recombinant Proteins / metabolism
Substrate Specificity
|
| IF |
5.915
|
| 引用数 |
27
|
|
WOS 分野
|
BIOCHEMISTRY & MOLECULAR BIOLOGY
|
| リソース情報 |
| 一般微生物 |
JCM6157 |
