Binding of steroid substrates reveals the key to the productive transition of the cytochrome P450 OleP

Antonella Costanzo; Francesca Fata; Ida Freda; Maria Laura De Sciscio; Elena Gugole; Giovanni Bulfaro; Matteo Di Renzo; Luca Barbizzi; Cécile Exertier; Giacomo Parisi; Marco D'Abramo; Beatrice Vallone; Carmelinda Savino; Linda Celeste Montemiglio

doi:10.1016/j.str.2024.06.005

Binding of steroid substrates reveals the key to the productive transition of the cytochrome P450 OleP

Structure. 2024 Sep 5;32(9):1465-1476.e3. doi: 10.1016/j.str.2024.06.005. Epub 2024 Jul 5.

Affiliations

¹ Department of Biochemical Sciences "Alessandro Rossi Fanelli", Sapienza, University of Rome, P. le Aldo Moro, 5, 00185 Rome, Italy; Takis Biotech, Via di Castel Romano 100, 00128 Rome, Italy.
² 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.
³ Department of Biochemical Sciences "Alessandro Rossi Fanelli", Sapienza, University of Rome, P. le Aldo Moro, 5, 00185 Rome, Italy.
⁴ Department of Chemistry, University of Rome, Sapienza, P.le A. Moro 5, 00185 Rome, Italy.
⁵ Department of Basic and Applied Sciences for Engineering (SBAI), Sapienza, University of Rome, Via Antonio Scarpa, 16, 00161 Rome, Italy.
⁶ Department of Biochemical Sciences "Alessandro Rossi Fanelli", Sapienza, University of Rome, P. le Aldo Moro, 5, 00185 Rome, Italy; 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. Electronic address: [email protected].
⁷ 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. Electronic address: [email protected].
⁸ 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. Electronic address: [email protected].

PMID: 38971159
DOI: 10.1016/j.str.2024.06.005

Abstract

OleP is a bacterial cytochrome P450 involved in oleandomycin biosynthesis as it catalyzes regioselective epoxidation on macrolide intermediates. OleP has recently been reported to convert lithocholic acid (LCA) into murideoxycholic acid through a highly regioselective reaction and to unspecifically hydroxylate testosterone (TES). Since LCA and TES mainly differ by the substituent group at the C17, here we used X-ray crystallography, equilibrium binding assays, and molecular dynamics simulations to investigate the molecular basis of the diverse reactivity observed with the two steroids. We found that the differences in the structure of TES and LCA affect the capability of these molecules to directly form hydrogen bonds with N-terminal residues of OleP internal helix I. The establishment of these contacts, by promoting the bending of helix I, fosters an efficient trigger of the open-to-closed structural transition that occurs upon substrate binding to OleP and contributes to the selectivity of the subsequent monooxygenation reaction.

Keywords: CYP107D1; OleP; X-ray crystallography; cytochrome P450; lithocholic acid; open-to-closed transition; selectivity; substrate binding; testosterone.

MeSH terms

Bacterial Proteins* / chemistry
Bacterial Proteins* / metabolism
Binding Sites
Crystallography, X-Ray
Cytochrome P-450 Enzyme System* / chemistry
Cytochrome P-450 Enzyme System* / metabolism
Hydrogen Bonding*
Hydroxylation
Lithocholic Acid / chemistry
Lithocholic Acid / metabolism
Molecular Dynamics Simulation*
Protein Binding*
Substrate Specificity
Testosterone* / chemistry
Testosterone* / metabolism

Substances

Cytochrome P-450 Enzyme System
Testosterone
Bacterial Proteins
Lithocholic Acid