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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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Cytochrome P450 OleP catalytic activity is strongly influenced by its structural dynamic conformational behavior. Here, we combine equilibrium-binding experiments with all-atom molecular dynamics simulations to clarify how different environments affect OleP conformational equilibrium between the open and the closed-catalytic competent-forms. Our data clearly show that at high-ionic strength conditions, the closed form is favored, and, very interestingly, different mechanisms, depending on the chemistry of the cations, can be used to rationalize such an effect.

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.

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.

The regulation of cytochrome P450 activity is often achieved by structural transitions induced by substrate binding. We describe the conformational transition experienced upon binding by the P450 OleP, an epoxygenase involved in oleandomycin biosynthesis. OleP bound to the substrate analog 6DEB crystallized in 2 forms: one with an ensemble of open and closed conformations in the asymmetric unit and another with only the closed conformation. Characterization of OleP-6DEB binding kinetics, also using the P450 inhibitor clotrimazole, unveiled a complex binding mechanism that involves slow conformational rearrangement with the accumulation of a spectroscopically detectable intermediate where 6DEB is bound to open OleP. Data reported herein provide structural snapshots of key precatalytic steps in the OleP reaction and explain how structural rearrangements induced by substrate binding regulate activity.-Parisi, G., Montemiglio, L. C., Giuffrè, A., Macone, A., Scaglione, A., Cerutti, G., Exertier, C., Savino, C., Vallone, B. Substrate-induced conformational change in cytochrome P450 OleP.

Background: OleP is a cyt P450 from Streptomyces antibioticus carrying out epoxigenation of the antibiotic oleandomycin during its biosynthesis. The timing of its reaction has not been fully clarified, doubts remain regarding its substrate and catalytic mechanism., Methods: The crystal structure of OleP in complex with clotrimazole, an inhibitor of P450s used in therapy, was solved and the complex formation dynamics was characterized by equilibrium and kinetic binding studies and compared to ketoconazole, another azole differing for the N1-substituent., Results: Clotrimazole coordinates the heme and occupies the active site. Most of the residues interacting with clotrimazole are conserved and involved in substrate binding in MycG, the P450 epoxigenase with the highest homology with OleP. Kinetic characterization of inhibitor binding revealed OleP to follow a simple bimolecular reaction, without detectable intermediates., Conclusions: Clotrimazole-bound OleP adopts an open form, held by a π-π stacking chain that fastens helices F and G and the FG loop. Affinity is affected by the interactions of the N1 substituent within the active site, given the one order of magnitude difference of the off-rate constants between clotrimazole and ketoconazole. Based on structural similarities with MycG, we propose a binding mode for both oleandomycin intermediates, that are the candidate substrates of OleP., General Significance: Among P450 epoxigenases OleP is the only one that introduces an epoxide on a non-activated C–C bond. The data here presented are necessary to understand the rare chemistry carried out by OleP, to engineer it and to design more selective and potent P450-targeted drugs.

The C-12 hydroxylase EryK is a bacterial cytochrome P450, active during one of the final tailoring steps of erythromycin A (ErA) biosynthesis. Its tight substrate specificity, restricted to the metabolic intermediate ErD, leads to the accumulation in the culture broth of a shunt metabolite, ErB, that originates from the competitive action of a methyltranferase on the substrate of EryK. Although the methylation of the mycarosyl moiety represents the only difference between the two metabolites, EryK exhibits very low conversion of ErB in ErA via a parallel pathway. Given its limited antimicrobial activity and its moderate toxicity, contamination by such a byproduct decreases the yield and purity of the antibiotic. In this study, EryK has been redesigned to make it suitable for industrial application. Taking advantage of the three-dimensional structure of the enzyme in complex with ErD, three single active-site mutants of EryK (M86A, H88E, and E89L) have been designed to allow hydroxylation of the nonphysiological substrate ErB. The binding and catalytic properties of these three variants on both ErD and ErB have been analyzed. Interestingly, we found the mutation of Met 86 to Ala to yield enzymatic activity on both ErB and ErD. The three-dimensional structure of the complex of mutated EryK with ErB revealed that the mutation allows ErB to accommodate in the active site of the enzyme and to induce its closure, thus assuring the progress of the catalytic reaction. Therefore, by single mutation the fine substrate recognition, active site closure, and locking were recovered.

EryK is a bacterial cytochrome P450 that catalyzes the last hydroxylation occurring during the biosynthetic pathway of erythromycin A in Streptomyces erythraeus. We report the crystal structures of EryK in complex with two widely used azole inhibitors: ketoconazole and clotrimazole. Both of these ligands use their imidazole moiety to coordinate the heme iron of P450s. Nevertheless, because of the different chemical and structural properties of their N1-substituent group, ketoconazole and clotrimazole trap EryK, respectively, in a closed and in an open conformation that resemble the two structures previously described for the ligand-free EryK. Indeed, ligands induce a distortion of the internal helix I that affects the accessibility of the binding pocket by regulating the kink of the external helix G via a network of interactions that involves helix F. The data presented thus constitute an example of how a cytochrome P450 may be selectively trapped in different conformational states by inhibitors.

Cytochrome P450s are heme-containing proteins that catalyze the oxidative metabolism of many physiological endogenous compounds. Because of their unique oxygen chemistry and their key role in drug and xenobiotic metabolism, particular attention has been devoted in elucidating their mechanism of substrate recognition. In this work, we analyzed the three-dimensional structures of a monomeric cytochrome P450 from Saccharopolyspora erythraea, commonly called EryK, and the binding kinetics to its physiological ligand, erythromycin D. Three different structures of EryK were obtained: two ligand-free forms and one in complex with its substrate. Analysis of the substrate-bound structure revealed the key structural determinants involved in substrate recognition and selectivity. Interestingly, the ligand-free structures of EryK suggested that the protein may explore an open and a closed conformation in the absence of substrate. In an effort to validate this hypothesis and to investigate the energetics between such alternative conformations, we performed stopped-flow absorbance experiments. Data demonstrated that EryK binds erythromycin D via a mechanism involving at least two steps. Contrary to previously characterized cytochrome P450s, analysis of double jump mixing experiments confirmed that this complex scenario arises from a pre-existing equilibrium between the open and closed subpopulations of EryK, rather than from an induced-fit type mechanism.

Erythromycin A is produced by Saccharopolyspora erythraea via a secondary metabolic pathway using several steps including glycosylations and hydroxylations of the first macrolide intermediate 6-deoxyerythronolide B. Erythromycin C-12 hydroxylase (CYP113A1), the P450 cytochrome active in the final stages of erythromycin biosynthesis, was cloned and expressed in E. coli. Different crystal forms were harvested from distinct crystallization conditions: two ligand-free forms, one substrate bound and two inhibitors-bound. All crystals belong either to the monoclinc P2(1)or to the orthorhombic P2(1)2(1)2(1) space groups, and exhibit diffraction limits ranging from 2.3 to 1.6 A. The structures will be determined by molecular replacement.