TY - JOUR
T1 - Phanerochaete chrysosporium NADPH-cytochrome P450 reductase kinetic mechanism
AU - Kelly, Steven Lewis
AU - Lamb, David Christopher
AU - Warrilow, Andrew G. S.
AU - Kelly, Diane Elizabeth
N1 - Warrilow, A. G. S., Lamb, D. C., Kelly, D. E., Kelly, S. L. (2002). Phanerochaete chrysosporium NADPH-cytochrome P450 reductase kinetic mechanism. Biochemical and Biophysical Research Communications, 299, (2), 189-195
Sponsorship: BBSRC
PY - 2002/11/29
Y1 - 2002/11/29
N2 - The recently completed genome of the basidiomycete, Phanerochaete chrysosporium, revealed the presence of one NADPH-cytochrome P450 oxidoreductase (CPR; EC 1.6.2.4) gene and >123 cytochrome P450 (CYP) genes. How a single CPR can drive many CYPs is an important area of study. We have investigated this CPR to gain insight into the mechanistic and structural biodiversity of the cytochrome P450 catalytic system. Native CPR and a NH2-terminally truncated derivative lacking 23 amino acids have been overexpressed in Escherichia coli and purified to electrophoretic homogeneity. Steady-state kinetics of cytochrome c reductase activity revealed a random sequential bireactant kinetic mechanism in which both products form dead-end complexes reflecting differences in CPR kinetic mechanisms even within a single kingdom of life. Removal of the N-terminal anchor of P. chrysosporium CPR did not alter the kinetic properties displayed by the enzyme in vitro, indicating it was a useful modification for structural studies.
AB - The recently completed genome of the basidiomycete, Phanerochaete chrysosporium, revealed the presence of one NADPH-cytochrome P450 oxidoreductase (CPR; EC 1.6.2.4) gene and >123 cytochrome P450 (CYP) genes. How a single CPR can drive many CYPs is an important area of study. We have investigated this CPR to gain insight into the mechanistic and structural biodiversity of the cytochrome P450 catalytic system. Native CPR and a NH2-terminally truncated derivative lacking 23 amino acids have been overexpressed in Escherichia coli and purified to electrophoretic homogeneity. Steady-state kinetics of cytochrome c reductase activity revealed a random sequential bireactant kinetic mechanism in which both products form dead-end complexes reflecting differences in CPR kinetic mechanisms even within a single kingdom of life. Removal of the N-terminal anchor of P. chrysosporium CPR did not alter the kinetic properties displayed by the enzyme in vitro, indicating it was a useful modification for structural studies.
KW - NADPH-cytochrome P450 oxidoreductase
KW - Cytochrome P450
KW - Phanerochaete chrysosporium
KW - Purification
KW - Kinetics
KW - Reaction mechanism
U2 - 10.1016/S0006-291X(02)02600-1
DO - 10.1016/S0006-291X(02)02600-1
M3 - Article
SN - 1090-2104
VL - 299
SP - 189
EP - 195
JO - Biochemical and Biophysical Research Communications
JF - Biochemical and Biophysical Research Communications
IS - 2
ER -