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Dissecting the Cytochrome P450 OleP Substrate Specificity: Evidence for a Preferential Substrate

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...

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Autores principales: Parisi, Giacomo, Freda, Ida, Exertier, Cécile, Cecchetti, Cristina, Gugole, Elena, Cerutti, Gabriele, D’Auria, Lucia, Macone, Alberto, Vallone, Beatrice, Savino, Carmelinda, Montemiglio, Linda Celeste
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7600006/
https://www.ncbi.nlm.nih.gov/pubmed/33036250
http://dx.doi.org/10.3390/biom10101411
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author Parisi, Giacomo
Freda, Ida
Exertier, Cécile
Cecchetti, Cristina
Gugole, Elena
Cerutti, Gabriele
D’Auria, Lucia
Macone, Alberto
Vallone, Beatrice
Savino, Carmelinda
Montemiglio, Linda Celeste
author_facet Parisi, Giacomo
Freda, Ida
Exertier, Cécile
Cecchetti, Cristina
Gugole, Elena
Cerutti, Gabriele
D’Auria, Lucia
Macone, Alberto
Vallone, Beatrice
Savino, Carmelinda
Montemiglio, Linda Celeste
author_sort Parisi, Giacomo
collection PubMed
description 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.
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spelling pubmed-76000062020-11-01 Dissecting the Cytochrome P450 OleP Substrate Specificity: Evidence for a Preferential Substrate Parisi, Giacomo Freda, Ida Exertier, Cécile Cecchetti, Cristina Gugole, Elena Cerutti, Gabriele D’Auria, Lucia Macone, Alberto Vallone, Beatrice Savino, Carmelinda Montemiglio, Linda Celeste Biomolecules Article 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. MDPI 2020-10-06 /pmc/articles/PMC7600006/ /pubmed/33036250 http://dx.doi.org/10.3390/biom10101411 Text en © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Parisi, Giacomo
Freda, Ida
Exertier, Cécile
Cecchetti, Cristina
Gugole, Elena
Cerutti, Gabriele
D’Auria, Lucia
Macone, Alberto
Vallone, Beatrice
Savino, Carmelinda
Montemiglio, Linda Celeste
Dissecting the Cytochrome P450 OleP Substrate Specificity: Evidence for a Preferential Substrate
title Dissecting the Cytochrome P450 OleP Substrate Specificity: Evidence for a Preferential Substrate
title_full Dissecting the Cytochrome P450 OleP Substrate Specificity: Evidence for a Preferential Substrate
title_fullStr Dissecting the Cytochrome P450 OleP Substrate Specificity: Evidence for a Preferential Substrate
title_full_unstemmed Dissecting the Cytochrome P450 OleP Substrate Specificity: Evidence for a Preferential Substrate
title_short Dissecting the Cytochrome P450 OleP Substrate Specificity: Evidence for a Preferential Substrate
title_sort dissecting the cytochrome p450 olep substrate specificity: evidence for a preferential substrate
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7600006/
https://www.ncbi.nlm.nih.gov/pubmed/33036250
http://dx.doi.org/10.3390/biom10101411
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