TY - JOUR
T1 - Structural and functional characterization of "laboratory evolved" cytochrome P450cam mutants showing enhanced naphthalene oxygenation activity
AU - Matsuura, Koji
AU - Tosha, Takehiko
AU - Yoshioka, Shiro
AU - Takahashi, Satoshi
AU - Ishimori, Koichiro
AU - Morishima, Isao
N1 - Funding Information:
This work was supported by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology in Japan (12002008 to I.M.; 14658217 to K.I.).
PY - 2004/10/29
Y1 - 2004/10/29
N2 - To elucidate molecular mechanisms for the enhanced oxygenation activity in the three mutants of cytochrome P450cam screened by 'laboratory evolution' [Nature 399 (1999) 670], we purified the mutants and characterized their functional and structural properties. The electronic absorption and resonance Raman spectra revealed that the structures of heme binding site of all purified mutants were quite similar to that of the wild-type enzyme, although the fraction of the inactivated form, called "P420," was increased. In the reaction with H 2O 2, only trace amounts of the naphthalene hydroxylation product were detected by gas chromatography. We, therefore, conclude that the three mutants do not exhibit significant changes in the structural and functional properties from those of wild-type P450cam except for the stability of the axial ligand in the reduced form. The enhanced fluorescence in the whole-cell assay would reflect enhancement in the oxygenation activity below the detectable limit of the gas chromatography and/or contributions of other reactions catalyzed by the heme iron.
AB - To elucidate molecular mechanisms for the enhanced oxygenation activity in the three mutants of cytochrome P450cam screened by 'laboratory evolution' [Nature 399 (1999) 670], we purified the mutants and characterized their functional and structural properties. The electronic absorption and resonance Raman spectra revealed that the structures of heme binding site of all purified mutants were quite similar to that of the wild-type enzyme, although the fraction of the inactivated form, called "P420," was increased. In the reaction with H 2O 2, only trace amounts of the naphthalene hydroxylation product were detected by gas chromatography. We, therefore, conclude that the three mutants do not exhibit significant changes in the structural and functional properties from those of wild-type P450cam except for the stability of the axial ligand in the reduced form. The enhanced fluorescence in the whole-cell assay would reflect enhancement in the oxygenation activity below the detectable limit of the gas chromatography and/or contributions of other reactions catalyzed by the heme iron.
KW - C-H bond activation
KW - Cytochrome P450
KW - Dioxygen
KW - Electronic absorption
KW - Gas chromatographic analysis
KW - Hydrogen peroxide
KW - Laboratory evolution
KW - Naphthalene oxidation
KW - P420 form
KW - Resonance Raman
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U2 - 10.1016/j.bbrc.2004.08.221
DO - 10.1016/j.bbrc.2004.08.221
M3 - Article
C2 - 15451425
AN - SCOPUS:4644299673
VL - 323
SP - 1209
EP - 1215
JO - Biochemical and Biophysical Research Communications
JF - Biochemical and Biophysical Research Communications
SN - 0006-291X
IS - 4
ER -