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Allosteric feedback inhibition of pyridoxine 5'-phosphate oxidase from Escherichia coli.

ABSTRACT: 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. PNPOx has been proposed to undergo product inhibition resulting from PLP binding at the active site. PLP has 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 observations open up new horizons on the mechanisms that 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.

SUBMITTER: Barile A

PROVIDER: S-EPMC6816108 | biostudies-literature | 2019 Oct

REPOSITORIES: biostudies-literature

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Allosteric feedback inhibition of pyridoxine 5'-phosphate oxidase from Escherichia coli.

Barile Anna A Tramonti Angela A di Salvo Martino Luigi ML Nogués Isabel I Nardella Caterina C Malatesta Francesco F Contestabile Roberto R

The Journal of biological chemistry 20190904 43

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. PNPOx has been proposed to undergo product inhibition resultin ...[more]

PMID: 31484724

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Project description:Escherichia coli YggS (COG0325) is a member of the highly conserved pyridoxal 5'-phosphate (PLP)-binding protein (PLPBP) family. Recent studies suggested a role for this protein family in the homeostasis of vitamin B6 and amino acids. The deletion or mutation of a member of this protein family causes pleiotropic effects in many organisms and is causative of vitamin B6-dependent epilepsy in humans. To date, little has been known about the mechanism by which lack of YggS results in these diverse phenotypes. In this study, we determined that the pyridoxine (PN) sensitivity observed in yggS-deficient E. coli was caused by the pyridoxine 5'-phosphate (PNP)-dependent overproduction of Val, which is toxic to E. coli The data suggest that the yggS mutation impacts Val accumulation by perturbing the biosynthetic of Thr from homoserine (Hse). Exogenous Hse inhibited the growth of the yggS mutant, caused further accumulation of PNP, and increased the levels of some intermediates in the Thr-Ile-Val metabolic pathways. Blocking the Thr biosynthetic pathway or decreasing the intracellular PNP levels abolished the perturbations of amino acid metabolism caused by the exogenous PN and Hse. Our data showed that a high concentration of intracellular PNP is the root cause of at least some of the pleiotropic phenotypes described for a yggS mutant of E. coliIMPORTANCE Recent studies showed that deletion or mutation of members of the YggS protein family causes pleiotropic effects in many organisms. Little is known about the causes, mechanisms, and consequences of these diverse phenotypes. It was previously shown that yggS mutations in E. coli result in the accumulation of PNP and some metabolites in the Ile/Val biosynthetic pathway. This work revealed that some exogenous stresses increase the aberrant accumulation of PNP in the yggS mutant. In addition, the current report provides evidence indicating that some, but not all, of the phenotypes of the yggS mutant in E. coli are due to the elevated PNP level. These results will contribute to continuing efforts to determine the molecular functions of the members of the YggS protein family.

Dataset Information

Allosteric feedback inhibition of pyridoxine 5'-phosphate oxidase from Escherichia coli.

Publications

Allosteric feedback inhibition of pyridoxine 5'-phosphate oxidase from <i>Escherichia coli</i>.

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