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 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.

The Oxidation of Hydrophobic Aromatic Substrates by Using a Variant of the P450 Monooxygenase CYP101B1

“…For most of the hydrophobic compounds tested, the NADH oxidation rate increased with the mutated form, in line with the greater spin‐state shift; this suggests that substrate binding plays a role in gating the beginning of the catalytic cycle . The rates of product formation for some substrates were relatively high, despite inducing lower spin‐state shifts; this suggest that the gating mechanism in CYP101B1 might be less stringent than is observed in other systems such as CYP101A1, and is in agreement with results obtained for other CYP101 family enzymes . When incorporated into a whole‐cell biotransformation system with the physiological electron‐transfer partners ArR/Arx, the CYP101B1 H85F variant was capable of synthesising the oxidised metabolites of alkylbenzenes and naphthalenes in good yield for characterisation (18–33 mg of purified naphthalene metabolites were generated from 200 mL of culture after a 16‐h reaction performed as described in the Experimental Section).…”

Recent Structural Insights into Cytochrome P450 Function

“…In the open structure of the camphor complex, the substrate shields the oxygen binding site from the solvent in the entrance channel, and it may not be necessary for the protein to close for efficient metabolism of camphor. However, some substrate induced-displacements seen in residual dipolar coupling-NMR have not been seen in crystal structures of substrate-free and -bound P450 cam [46]. The P450 cam structure may be free to sample conformational space in ways that are prevented by crystallographic packing constraints.…”

“…Will there be breakthroughs in the development of new higher throughput crystallography technology? Can NMR systems such as residual dipolar coupling be applied after crystal structures are solved [46, 118]? Will we find that most P450s can be defined in the context of a small number of different states that can be associated with all ligands, and can we utilize this in modeling?…”

The Metal Drives the Chemistry: Dual Functions of Acireductone Dioxygenase

“…It is becoming clear that not just the active site, but the entire enzyme structure is involved in this control. 124 As such, re-engineering to improve or modify an enzyme to reach high efficiencies must take this requirement into account. The most straightforward way of mimicking natural selection is to use directed evolution, with iterative rounds of enzyme diversification and functional screening.…”