In Escherichia coli, the synthesis of pyridoxal 5'-phosphate (PLP), the catalytically active form of vitamin B6, takes place through the so-called deoxyxylulose 5-phosphate-dependent pathway, whose last step is pyridoxine 5'-phosphate (PNP) oxidation to PLP, catalyzed by the FMN-dependent enzyme PNP oxidase (PNPOx). This enzyme plays a pivotal role in controlling intracellular homeostasis and bioavailability of PLP.PNPOxhas been proposed to undergo product inhibition resulting from PLPbinding at the active site.PLPhas also been reported to bind tightly at a secondary site, apparently without causing PNPOx inhibition. The possible location of this secondary site has been indicated by crystallographic studies as two symmetric surface pockets present on the PNPOx homodimer, but this site has never been verified by other experimental means. Here, we demonstrate, through kinetic measurements, that PLP inhibition is actually of a mixed-type nature and results from binding of this vitamer at an allosteric site. This interpretation was confirmed by the characterization of a mutated PNPOx form, in which substrate binding at the active site is heavily hampered but PLP binding is preserved. Structural and functional connections between the active site and the allosteric site were indicated by equilibrium binding experiments, which revealed different PLP-binding stoichiometries with WT and mutant PNPOx forms. These observationsopenupnewhorizonsonthemechanismsthat regulate E. coli PNPOx, which may have commonalities with the mechanisms regulating human PNPOx, whose crucial role in vitamin B6 metabolism and epilepsy is well-known.
Allosteric feedback inhibition of pyridoxine 5'-phosphate oxidase from escherichia coli / Barile, A.; Tramonti, A.; Di Salvo, M. L.; Nogues, I.; Nardella, C.; Malatesta, F.; Contestabile, R.. - In: THE JOURNAL OF BIOLOGICAL CHEMISTRY. - ISSN 0021-9258. - 294:43(2019), pp. 15593-15603.
Scheda prodotto non validato
Attenzione! I dati visualizzati non sono stati sottoposti a validazione da parte dell'ateneo
|Titolo:||Allosteric feedback inhibition of pyridoxine 5'-phosphate oxidase from escherichia coli|
CONTESTABILE, Roberto (Corresponding author)
|Data di pubblicazione:||2019|
|Citazione:||Allosteric feedback inhibition of pyridoxine 5'-phosphate oxidase from escherichia coli / Barile, A.; Tramonti, A.; Di Salvo, M. L.; Nogues, I.; Nardella, C.; Malatesta, F.; Contestabile, R.. - In: THE JOURNAL OF BIOLOGICAL CHEMISTRY. - ISSN 0021-9258. - 294:43(2019), pp. 15593-15603.|
|Appartiene alla tipologia:||01a Articolo in rivista|