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

Barile, Anna; Battista, Theo; Fiorillo, Annarita; Salvo, Martino L. di; Malatesta, Francesco; Tramonti, Angela; Ilari, Andrea; Contestabile, Roberto

doi:10.1016/j.jbc.2021.100795

Cited by 11 publications

(23 citation statements)

References 18 publications

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“…The ensuing restoration of the impaired PLP transfer in E144K CBS with the PLP-modified K144 may explain the therapeutic significance of B6 administration in this mutant, absent in the T191M mutant. Another mechanism contributing to the patients’ response to B6 therapy upon mutations affecting the heterologous interactions may be allosteric regulation of the PLP producers by their substrates and products [17, 49], potentially affecting also the heterologous interactions with PLP acceptors.…”

Section: Resultsmentioning

confidence: 99%

“…For instance, at 0.5 mM, these vitamers are much more effective in killing varous cancer cells, than pyridoxamine or pyridoxin [16]. In view of the PLP reactivity, it has been hypothesized that PLP is transferred directly from its donors to the enzymes which use PLP as the coenzyme (PLP acceptors) [17][18][19]. However, the structurally different proteins employing PLP as their coenzyme, encompass up to seven structural folds without significant homology [1,20].…”

Section: Introductionmentioning

confidence: 99%

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Protein-protein interfaces as druggable targets: A common motif of the pyridoxal-5’-phosphate-dependent enzymes to receive the coenzyme from its producers

Aleshin¹,

Bunik

2023

Preprint

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Pyridoxal-5'-phosphate (PLP), a phosphorylated form of vitamin B6, acts as a coenzyme for numerous reactions, including those changed in cancer and/or associated with the disease prognosis. Since highly reactive PLP may modify cellular proteins, it is hypothesized to be directly transferred from its donors to acceptors. Our goal is to validate the hypothesis by finding common motif(s) in a multitude of the PLP-dependent enzymes for binding the limited number of the PLP donors, namely pyridoxal kinase (PdxK), pyridox(am)in-5'-phosphate oxidase (PNPO) and the PLP-binding protein (PLPBP). Experimentally confirmed interactions between the PLP donors and acceptors reveal that PdxK and PNPO interact with the PLP acceptors of folds I and II, while PLPBP - with those of folds III and V. Aligning the sequences and 3D structures of the identified interactors of PdxK and PNPO, we have found a common motif in the PLP-dependent enzymes of folds I and II. The motif extends from the enzyme surface to the neighborhood of the PLP binding site, represented by an exposed alfa-helix, a partially buried beta-strand and residual loops. Pathogenicity of mutations in human PLP-dependent enzymes within or in the vicinity of the motif, but outside of the active sites, supports functional significance of the motif that may provide an interface for the direct transfer of PLP from the sites of its synthesis to those of the coenzyme binding. The enzyme-specific amino acid residues of the common motif may be useful to develop selective inhibitors blocking PLP delivery to the PLP-dependent enzymes critical for proliferation of malignant cells.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Protein-protein interfaces as druggable targets: A common motif of the pyridoxal-5’-phosphate-dependent enzymes to receive the coenzyme from its producers

Aleshin¹,

Bunik

2023

Preprint

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“…All these observations, combined with the inefficient action of PdxY on PL salvage outlined by our experiments, explain the prevalence of the longer route of PL salvage involving PdxI under PL supplementation. We have shown that PdxH activity is under fine allosteric control by PLP acting as a retroactive inhibitor [44,45]. From this point of view, directing PL towards PN and thus the action of PdxH could be a way to limit the accumulation of PL and at the same time regulate PLP formation.…”

Section: Discussionmentioning

confidence: 99%

Functional and structural properties of pyridoxal reductase (PdxI) from Escherichia coli: a pivotal enzyme in the vitamin B6 salvage pathway

Tramonti,

Donkor,

Parroni

et al. 2023

The FEBS Journal

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Pyridoxine 4‐dehydrogenase (PdxI), a NADPH‐dependent pyridoxal reductase, is one of the key players in the Escherichia coli pyridoxal 5’‐phosphate (PLP) salvage pathway. This enzyme, which catalyses the reduction of pyridoxal into pyridoxine, causes pyridoxal to be converted into PLP via the formation of pyridoxine and pyridoxine phosphate. The structural and functional properties of PdxI were hitherto unknown, preventing a rational explanation of how and why this longer, detoured pathway occurs, given that, in E. coli, two pyridoxal kinases (PdxK and PdxY) exist that could convert pyridoxal directly into PLP. Here, we report a detailed characterisation of E. coli PdxI that explains this behaviour. The enzyme efficiently catalyses the reversible transformation of pyridoxal into pyridoxine, although the reduction direction is thermodynamically strongly favoured, following a compulsory‐order ternary‐complex mechanism. In vitro, the enzyme is also able to catalyse PLP reduction and use NADH as an electron donor, although with lower efficiency. As with all members of the aldo‐keto reductase (AKR) superfamily, the enzyme has a TIM barrel fold; however, it shows some specific features, the most important of which is the presence of an Arg residue that replaces the catalytic tetrad His residue that is present in all AKRs and appears to be involved in substrate specificity. The above results, in conjunction with kinetic and static measurements of vitamins B6 in cell extracts of E. coli wild‐type and knockout strains, shed light on the role of PdxI and both kinases in determining the pathway followed by pyridoxal in its conversion to PLP, which has a precise regulatory function.

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“…In microorganisms, like Escherichia coli, which synthesize PLP through the socalled de novo DXP-dependent pathway, the PNPO reaction represents the last irreplaceable step of PLP biosynthesis (Tramonti et al, 2021). In both human and E. coli PNPO, PLP works as a regulator of its own production because it binds at an allosteric site of the enzyme, affecting the binding of substrate at the active site and inhibiting the catalytic activity (Barile et al, 2019(Barile et al, , 2021. This shows that a cross-talk between the allosteric site and the active site is present.…”

Section: Introductionmentioning

confidence: 99%

Identification of the pyridoxal 5′‐phosphate allosteric site in human pyridox(am)ine 5′‐phosphate oxidase

Barile,

Graziani,

Antonelli

et al. 2024

Protein Science

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Adequate levels of pyridoxal 5′‐phosphate (PLP), the catalytically active form of vitamin B6, and its proper distribution in the body are essential for human health. The PLP recycling pathway plays a crucial role in these processes and its defects cause severe neurological diseases. The enzyme pyridox(am)ine 5′‐phosphate oxidase (PNPO), whose catalytic action yields PLP, is one of the key players in this pathway. Mutations in the gene encoding PNPO are responsible for a severe form of neonatal epilepsy. Recently, PNPO has also been described as a potential target for chemotherapeutic agents. Our laboratory has highlighted the crucial role of PNPO in the regulation of PLP levels in the cell, which occurs via a feedback inhibition mechanism of the enzyme, exerted by binding of PLP at an allosteric site. Through docking analyses and site‐directed mutagenesis experiments, here we identified the allosteric PLP binding site of human PNPO. This site is located in the same protein region as the allosteric site we previously identified in the Escherichia coli enzyme homologue. However, the identity and arrangement of the amino acid residues involved in PLP binding are completely different and resemble those of the active site of PLP‐dependent enzymes. The identification of the PLP allosteric site of human PNPO paves the way for the rational design of enzyme inhibitors as potential anti‐cancer compounds.

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

Cited by 11 publications

References 18 publications

Protein-protein interfaces as druggable targets: A common motif of the pyridoxal-5’-phosphate-dependent enzymes to receive the coenzyme from its producers

Protein-protein interfaces as druggable targets: A common motif of the pyridoxal-5’-phosphate-dependent enzymes to receive the coenzyme from its producers

Functional and structural properties of pyridoxal reductase (PdxI) from Escherichia coli: a pivotal enzyme in the vitamin B6 salvage pathway

Identification of the pyridoxal 5′‐phosphate allosteric site in human pyridox(am)ine 5′‐phosphate oxidase

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