title: Detection of substrate-dependent conformational changes in the P450 fold by nuclear magnetic resonance.
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 P450(cam) (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.
authors.affiliation: Departments of Chemistry and Biochemistry Brandeis University, 415 South St., Waltham MA 02454-9110, USA.
authors.forename: Allison M
authors.initials: AM
authors.lastname: Colthart
authors.affiliation: Departments of Chemistry and Biochemistry Brandeis University, 415 South St., Waltham MA 02454-9110, USA.
authors.forename: Drew R
authors.initials: DR
authors.lastname: Tietz
authors.affiliation: Departments of Chemistry and Biochemistry Brandeis University, 415 South St., Waltham MA 02454-9110, USA.
authors.forename: Yuhua
authors.initials: Y
authors.lastname: Ni
authors.affiliation: Departments of Chemistry and Biochemistry Brandeis University, 415 South St., Waltham MA 02454-9110, USA.
authors.forename: Jessica L
authors.initials: JL
authors.lastname: Friedman
authors.affiliation: Departments of Chemistry and Biochemistry Brandeis University, 415 South St., Waltham MA 02454-9110, USA.
authors.forename: Marina
authors.initials: M
authors.lastname: Dang
authors.affiliation: Departments of Chemistry and Biochemistry Brandeis University, 415 South St., Waltham MA 02454-9110, USA.
authors.forename: Thomas C
authors.initials: TC
authors.lastname: Pochapsky
mesh_terms: D001665:Binding Sites; D019475:Camphor 5-Monooxygenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D003577:Cytochrome P-450 Enzyme System; D019475:Camphor 5-Monooxygenase