Abstract
Cytochrome P450 monooxygenases typically catalyze the insertion of one atom of oxygen from O2 into unactivated carbon-hydrogen and carbon-carbon bonds, with concomitant reduction of the other oxygen atom to H2O by NAD(P)H. Comparison of the average structures of the camphor hydroxylase cytochrome P450cam (CYP101) obtained from residual dipolar coupling (RDC)-restrained molecular dynamics (MD) in the presence and absence of substrate camphor shows structural displacements resulting from the essential collapse of the active site upon substrate removal. This collapse has conformational consequences that extend across the protein structure, none of which were observed in analogous crystallographic structures. Mutations were made to test the involvement of the observed conformational changes in substrate binding and recognition. All of the mutations performed based upon the NMR-detected perturbations, even those remote from the active site, resulted in modified substrate selectivity, enzyme efficiency and/or haem iron spin state. The results demonstrate that solution NMR can provide insights into enzyme structure-function relationships that are difficult to obtain by other methods.
Highlights
MethodsOverexpression and purification of CYP101 for enzymatic assays
We made extensive sequential resonance assignments for CYP101 using multidimensional nuclear magnetic resonance (NMR) methods[12,13]. These assignments have given us unprecedented insight into local structure/function relationships in this enzyme, and we recently described the use of residual dipolar coupling (RDC) to generate structural ensembles of in both the camphor-bound and substrate free forms of CYP1019,10
The importance of the current work is twofold: First, we demonstrate that high-resolution solution NMR techniques applied to complex enzymes can provide detailed and functionally relevant structural information that has not been obtained to date by other methods
Summary
Overexpression and purification of CYP101 for enzymatic assays. Plasmid pDNC334A encoding the gene for CYP101 C334A was transformed into Escherichia coli NCM533 cells by electroporation. The C334A mutant of CYP101 is spectroscopically and enzymatically identical to wild-type (WT) and is referred to as such. Protein expression was induced with the addition of IPTG to a final concentration of 1 mM. Expression was carried out at 28° for 18 hours. Cells were pelleted by centrifugation at 2220 × g at 4 °C. Pellets were resuspended in 50 mM Tris·HCl, 50 mM KCl, pH 7.4. The pellet was discarded and the supernatant was filtered through a 0.45 μ m filter and applied to a DEAE Sepharose column (GE Healthcare) pre-equilibrated with 50 mM Tris·HCl, 50 mM KCl, pH 7.4. After elution with a linear gradient of 50 mM KCl to 300 mM KCl, fractions with A417/A280 > 0.4 were combined and used for assays
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