Dissecting the Cytochrome P450 OleP Substrate Specificity: Evidence for a Preferential Substrate
Giacomo Parisi,
Ida Freda,
Cécile Exertier,
Cristina Cecchetti,
Elena Gugole,
Gabriele Cerutti,
Lucia D’Auria,
Alberto Macone,
Beatrice Vallone,
Carmelinda Savino,
Linda Celeste Montemiglio
Affiliations
Giacomo Parisi
Istituto Pasteur-Fondazione Cenci Bolognetti and Department of Biochemical Sciences “Alessandro Rossi Fanelli”, Sapienza, University of Rome, P. le Aldo Moro, 5, 00185 Rome, Italy
Ida Freda
Istituto Pasteur-Fondazione Cenci Bolognetti and Department of Biochemical Sciences “Alessandro Rossi Fanelli”, Sapienza, University of Rome, P. le Aldo Moro, 5, 00185 Rome, Italy
Cécile Exertier
Istituto Pasteur-Fondazione Cenci Bolognetti and Department of Biochemical Sciences “Alessandro Rossi Fanelli”, Sapienza, University of Rome, P. le Aldo Moro, 5, 00185 Rome, Italy
Cristina Cecchetti
Istituto Pasteur-Fondazione Cenci Bolognetti and Department of Biochemical Sciences “Alessandro Rossi Fanelli”, Sapienza, University of Rome, P. le Aldo Moro, 5, 00185 Rome, Italy
Elena Gugole
Istituto Pasteur-Fondazione Cenci Bolognetti and Department of Biochemical Sciences “Alessandro Rossi Fanelli”, Sapienza, University of Rome, P. le Aldo Moro, 5, 00185 Rome, Italy
Gabriele Cerutti
Istituto Pasteur-Fondazione Cenci Bolognetti and Department of Biochemical Sciences “Alessandro Rossi Fanelli”, Sapienza, University of Rome, P. le Aldo Moro, 5, 00185 Rome, Italy
Lucia D’Auria
Department of Biochemical Sciences “Alessandro Rossi Fanelli”, Sapienza, University of Rome, P. le Aldo Moro, 5, 00185 Rome, Italy
Alberto Macone
Department of Biochemical Sciences “Alessandro Rossi Fanelli”, Sapienza, University of Rome, P. le Aldo Moro, 5, 00185 Rome, Italy
Beatrice Vallone
Istituto Pasteur-Fondazione Cenci Bolognetti and Department of Biochemical Sciences “Alessandro Rossi Fanelli”, Sapienza, University of Rome, P. le Aldo Moro, 5, 00185 Rome, Italy
Carmelinda Savino
Institute of Molecular Biology and Pathology c/o Department of Biochemical Sciences “Alessandro Rossi Fanelli”, Sapienza, University of Rome, National Research Council, P.le Aldo Moro, 5, 00185 Rome, Italy
Linda Celeste Montemiglio
Institute of Molecular Biology and Pathology c/o Department of Biochemical Sciences “Alessandro Rossi Fanelli”, Sapienza, University of Rome, National Research Council, P.le Aldo Moro, 5, 00185 Rome, Italy
The cytochrome P450 OleP catalyzes the epoxidation of aliphatic carbons on both the aglycone 8.8a-deoxyoleandolide (DEO) and the monoglycosylated L-olivosyl-8.8a-deoxyoleandolide (L-O-DEO) intermediates of oleandomycin biosynthesis. We investigated the substrate versatility of the enzyme. X-ray and equilibrium binding data show that the aglycone DEO loosely fits the OleP active site, triggering the closure that prepares it for catalysis only on a minor population of enzyme. The open-to-closed state transition allows solvent molecules to accumulate in a cavity that forms upon closure, mediating protein–substrate interactions. In silico docking of the monoglycosylated L-O-DEO in the closed OleP–DEO structure shows that the L-olivosyl moiety can be hosted in the same cavity, replacing solvent molecules and directly contacting structural elements involved in the transition. X-ray structures of aglycone-bound OleP in the presence of L-rhamnose confirm the cavity as a potential site for sugar binding. All considered, we propose L-O-DEO as the optimal substrate of OleP, the L-olivosyl moiety possibly representing the molecular wedge that triggers a more efficient structural response upon substrate binding, favoring and stabilizing the enzyme closure before catalysis. OleP substrate versatility is supported by structural solvent molecules that compensate for the absence of a glycosyl unit when the aglycone is bound.
