Volume 15, Issue 6, Pages (June 2008)

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Volume 15, Issue 6, Pages 563-572 (June 2008) 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 Chemistry & Biology Volume 15, Issue 6, Pages 563-572 (June 2008) DOI: 10.1016/j.chembiol.2008.05.013 Copyright © 2008 Elsevier Ltd Terms and Conditions

Figure 1 CO-Reduced Difference Spectra of Cell-Free Extracts of B. subtilis CYPs (A) Solid line, CYP102A2; dotted line, CYP102A3; dashed line, CYP107H1. (B) Solid line, CYP109B1; dotted line, CYP107J1. (C) Solid line, CYP107K1; dotted line, CYP134A1; dashed line, CYP152A1. The protein concentrations were adjusted to 2 mg protein/ml. Concentrations of CYPs were calculated using the extinction coefficient and were as follows: CYP102A2, 0.21 μmol/g protein; CYP102A3, 0.22 μmol/g; CYP107H1, 0.33 μmol/g; CYP107J1, 1.3 μmol/g; CYP107K1, 0.40 μmol/g; CYP109B1, 1.4 μmol/g; CYP134A1, 0.12 μmol/g; CYP152A1, 0.35 μmol/g. Chemistry & Biology 2008 15, 563-572DOI: (10.1016/j.chembiol.2008.05.013) Copyright © 2008 Elsevier Ltd Terms and Conditions

Figure 2 FT-ICR/MS Analysis of the 30-Compound Mixture Open and closed triangles indicate [M+H]+ and [M+Na]+ peaks of the 30 compounds, respectively. Mass scale expansion for the position is indicated with an arrow (inset). Chemistry & Biology 2008 15, 563-572DOI: (10.1016/j.chembiol.2008.05.013) Copyright © 2008 Elsevier Ltd Terms and Conditions

Figure 3 FT-ICR/MS Analysis of the Monooxygenation of Compactin by CYP109B1 in the Compound Mixture Mass spectra after the reaction with CYP109B1 (+), without the enzyme (−), and with CYP109B1 under 18O2 atmosphere (+ under 18O2). Chemistry & Biology 2008 15, 563-572DOI: (10.1016/j.chembiol.2008.05.013) Copyright © 2008 Elsevier Ltd Terms and Conditions

Figure 4 HPLC-FT-ICR/MS Analysis of the CYP107J1 and CYP134A1 Reaction Products (A) HPLC chromatogram (254 nm) after the reaction of CYP107J1 with testosterone enanthate. The m/z value of each peak is also shown. (B) HPLC chromatogram after the reaction of CYP134A1 with progesterone. (C) MS/MS spectrum for the peak at 34.8 min in chromatogram (A). (D) MS/MS spectrum for the peak at 21.5 min in chromatogram (B). Chemistry & Biology 2008 15, 563-572DOI: (10.1016/j.chembiol.2008.05.013) Copyright © 2008 Elsevier Ltd Terms and Conditions