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Identification and characterization of the pyridoxal 5’-phosphate allosteric site in Escherichia coli pyridoxine 5’-phosphate oxidase

Pyridoxal 5’-phosphate (PLP), the catalytically active form of vitamin B(6), plays a pivotal role in metabolism as an enzyme cofactor. PLP is a very reactive molecule and can be very toxic unless its intracellular concentration is finely regulated. In Escherichia coli, PLP formation is catalyzed by...

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Autores principales: Barile, Anna, Battista, Theo, Fiorillo, Annarita, di Salvo, Martino Luigi, Malatesta, Francesco, Tramonti, Angela, Ilari, Andrea, Contestabile, Roberto
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Society for Biochemistry and Molecular Biology 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8215295/
https://www.ncbi.nlm.nih.gov/pubmed/34019876
http://dx.doi.org/10.1016/j.jbc.2021.100795
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author Barile, Anna
Battista, Theo
Fiorillo, Annarita
di Salvo, Martino Luigi
Malatesta, Francesco
Tramonti, Angela
Ilari, Andrea
Contestabile, Roberto
author_facet Barile, Anna
Battista, Theo
Fiorillo, Annarita
di Salvo, Martino Luigi
Malatesta, Francesco
Tramonti, Angela
Ilari, Andrea
Contestabile, Roberto
author_sort Barile, Anna
collection PubMed
description Pyridoxal 5’-phosphate (PLP), the catalytically active form of vitamin B(6), plays a pivotal role in metabolism as an enzyme cofactor. PLP is a very reactive molecule and can be very toxic unless its intracellular concentration is finely regulated. In Escherichia coli, PLP formation is catalyzed by pyridoxine 5’-phosphate oxidase (PNPO), a homodimeric FMN-dependent enzyme that is responsible for the last step of PLP biosynthesis and is also involved in the PLP salvage pathway. We have recently observed that E. coli PNPO undergoes an allosteric feedback inhibition by PLP, caused by a strong allosteric coupling between PLP binding at the allosteric site and substrate binding at the active site. Here we report the crystallographic identification of the PLP allosteric site, located at the interface between the enzyme subunits and mainly circumscribed by three arginine residues (Arg23, Arg24, and Arg215) that form an “arginine cage” and efficiently trap PLP. The crystal structure of the PNPO–PLP complex, characterized by a marked structural asymmetry, presents only one PLP molecule bound at the allosteric site of one monomer and sheds light on the allosteric inhibition mechanism that makes the enzyme-substrate–PLP ternary complex catalytically incompetent. Site-directed mutagenesis studies focused on the arginine cage validate the identity of the allosteric site and provide an effective means to modulate the allosteric properties of the enzyme, from the loosening of the allosteric coupling (in the R23L/R24L and R23L/R215L variants) to the complete loss of allosteric properties (in the R23L/R24L/R21L variant).
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spelling pubmed-82152952021-06-21 Identification and characterization of the pyridoxal 5’-phosphate allosteric site in Escherichia coli pyridoxine 5’-phosphate oxidase Barile, Anna Battista, Theo Fiorillo, Annarita di Salvo, Martino Luigi Malatesta, Francesco Tramonti, Angela Ilari, Andrea Contestabile, Roberto J Biol Chem Research Article Pyridoxal 5’-phosphate (PLP), the catalytically active form of vitamin B(6), plays a pivotal role in metabolism as an enzyme cofactor. PLP is a very reactive molecule and can be very toxic unless its intracellular concentration is finely regulated. In Escherichia coli, PLP formation is catalyzed by pyridoxine 5’-phosphate oxidase (PNPO), a homodimeric FMN-dependent enzyme that is responsible for the last step of PLP biosynthesis and is also involved in the PLP salvage pathway. We have recently observed that E. coli PNPO undergoes an allosteric feedback inhibition by PLP, caused by a strong allosteric coupling between PLP binding at the allosteric site and substrate binding at the active site. Here we report the crystallographic identification of the PLP allosteric site, located at the interface between the enzyme subunits and mainly circumscribed by three arginine residues (Arg23, Arg24, and Arg215) that form an “arginine cage” and efficiently trap PLP. The crystal structure of the PNPO–PLP complex, characterized by a marked structural asymmetry, presents only one PLP molecule bound at the allosteric site of one monomer and sheds light on the allosteric inhibition mechanism that makes the enzyme-substrate–PLP ternary complex catalytically incompetent. Site-directed mutagenesis studies focused on the arginine cage validate the identity of the allosteric site and provide an effective means to modulate the allosteric properties of the enzyme, from the loosening of the allosteric coupling (in the R23L/R24L and R23L/R215L variants) to the complete loss of allosteric properties (in the R23L/R24L/R21L variant). American Society for Biochemistry and Molecular Biology 2021-05-18 /pmc/articles/PMC8215295/ /pubmed/34019876 http://dx.doi.org/10.1016/j.jbc.2021.100795 Text en © 2021 The Authors https://creativecommons.org/licenses/by/4.0/This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Research Article
Barile, Anna
Battista, Theo
Fiorillo, Annarita
di Salvo, Martino Luigi
Malatesta, Francesco
Tramonti, Angela
Ilari, Andrea
Contestabile, Roberto
Identification and characterization of the pyridoxal 5’-phosphate allosteric site in Escherichia coli pyridoxine 5’-phosphate oxidase
title Identification and characterization of the pyridoxal 5’-phosphate allosteric site in Escherichia coli pyridoxine 5’-phosphate oxidase
title_full Identification and characterization of the pyridoxal 5’-phosphate allosteric site in Escherichia coli pyridoxine 5’-phosphate oxidase
title_fullStr Identification and characterization of the pyridoxal 5’-phosphate allosteric site in Escherichia coli pyridoxine 5’-phosphate oxidase
title_full_unstemmed Identification and characterization of the pyridoxal 5’-phosphate allosteric site in Escherichia coli pyridoxine 5’-phosphate oxidase
title_short Identification and characterization of the pyridoxal 5’-phosphate allosteric site in Escherichia coli pyridoxine 5’-phosphate oxidase
title_sort identification and characterization of the pyridoxal 5’-phosphate allosteric site in escherichia coli pyridoxine 5’-phosphate oxidase
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8215295/
https://www.ncbi.nlm.nih.gov/pubmed/34019876
http://dx.doi.org/10.1016/j.jbc.2021.100795
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