Abstract
Substrate binding to the cytochrome P450 OleP is coupled to a large open-to-closed transition that remodels the active site, minimizing its exposure to the external solvent. When the aglycone substrate binds, a small empty cavity is formed between the I and G helices, the BC loop, and the substrate itself, where solvent molecules accumulate mediating substrate-enzyme interactions. Herein, we analyzed the role of this cavity in substrate binding to OleP by producing three mutants (E89Y, G92W, and S240Y) to decrease its volume. The crystal structures of the OleP mutants in the closed state bound to the aglycone 6DEB showed that G92W and S240Y occupied the cavity, providing additional contact points with the substrate. Conversely, mutation E89Y induces a flipped-out conformation of this amino acid side chain, that points towards the bulk, increasing the empty volume. Equilibrium titrations and molecular dynamic simulations indicate that the presence of a bulky residue within the cavity impacts the binding properties of the enzyme, perturbing the conformational space explored by the complexes. Our data highlight the relevance of this region in OleP substrate binding and suggest that it represents a key substrate-protein contact site to consider in the perspective of redirecting its activity towards alternative compounds.
Highlights
To assess how mutations at the solvent cavity affect the functional properties of OleP, we performed equilibrium binding experiments using 6-deoxyerythronolide B (6DEB), monitoring the spin state shift of the heme iron that accompanies ligand binding to the P450 distal pocket
Binding of 6DEB to wild type OleP produces a low- to high-spin shift, which is responsible for the typical type I change of the low Soret absorption maximum from ~424 to ~387 nm in the the change in heme absorption was measured at 298 K as a function of substrate concentration for all of the mutants, while the enzyme concentration remained fixed
To the wild type, binding to E89Y causes a type I spectral change. This mutation marginally alters the affinity for the aglycone, but it halves the amplitude of the spectral transition, indicating the presence at the equilibrium of a minor population of enzyme, which is converted to the high spin state by 6DEB binding with respect to OleP
Summary
Substrate binding to OleP is coupled to a large structural transition, which originates from a small ensemble of short-range contacts established in the active site of the enzyme This activates an intramolecular cascade of interactions that affect the overall protein structure [7,8]. Mutants have been designed based on the previously reported structure of the closed aglycone-bound OleP, with the aim to decrease the volume of the cavity where the mutated residue could still accommodate their hydrophobic side chain, without hampering aglycone substrate binding. This was achieved by substitutions of E89 by tyrosine, G92 by tryptophan, and S240 by tyrosine. Heme and 6DEB are shown in red and khaki sticks, respectively
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