Point Mutations at a Key Site Alter the Cytochrome P450 OleP Structural Dynamics
Linda Celeste Montemiglio,
Elena Gugole,
Ida Freda,
Cécile Exertier,
Lucia D’Auria,
Cheng Giuseppe Chen,
Alessandro Nicola Nardi,
Gabriele Cerutti,
Giacomo Parisi,
Marco D’Abramo,
Carmelinda Savino,
Beatrice Vallone
Affiliations
Linda Celeste Montemiglio
Institute of Molecular Biology and Pathology, CNR c/o Department of Biochemical Sciences “A. Rossi Fanelli”, University of Rome, Sapienza, P.le A. Moro 5, 00185 Rome, Italy
Elena Gugole
Department of Biochemical Sciences “A. Rossi Fanelli”, University of Rome, Sapienza, P.le A. Moro 5, 00185 Rome, Italy
Ida Freda
Department of Biochemical Sciences “A. Rossi Fanelli”, University of Rome, Sapienza, P.le A. Moro 5, 00185 Rome, Italy
Cécile Exertier
Institute of Molecular Biology and Pathology, CNR c/o Department of Biochemical Sciences “A. Rossi Fanelli”, University of Rome, Sapienza, P.le A. Moro 5, 00185 Rome, Italy
Lucia D’Auria
Department of Biochemical Sciences “A. Rossi Fanelli”, University of Rome, Sapienza, P.le A. Moro 5, 00185 Rome, Italy
Cheng Giuseppe Chen
Department of Chemistry, University of Rome, Sapienza, P.le A. Moro 5, 00185 Rome, Italy
Alessandro Nicola Nardi
Department of Chemistry, University of Rome, Sapienza, P.le A. Moro 5, 00185 Rome, Italy
Gabriele Cerutti
Department of Biochemical Sciences “A. Rossi Fanelli”, University of Rome, Sapienza, P.le A. Moro 5, 00185 Rome, Italy
Giacomo Parisi
Center for Life Nano & Neuro-Science, Fondazione Istituto Italiano di Tecnologia, IIT, 00185 Rome, Italy
Marco D’Abramo
Department of Chemistry, University of Rome, Sapienza, P.le A. Moro 5, 00185 Rome, Italy
Carmelinda Savino
Institute of Molecular Biology and Pathology, CNR c/o Department of Biochemical Sciences “A. Rossi Fanelli”, University of Rome, Sapienza, P.le A. Moro 5, 00185 Rome, Italy
Beatrice Vallone
Department of Biochemical Sciences “A. Rossi Fanelli”, University of Rome, Sapienza, P.le A. Moro 5, 00185 Rome, Italy
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.
