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1.
Int J Mol Sci ; 25(6)2024 Mar 09.
Artigo em Inglês | MEDLINE | ID: mdl-38542149

RESUMO

Enzymes reliant on pyridoxal 5'-phosphate (PLP), the metabolically active form of vitamin B6, hold significant importance in both biology and medicine. They facilitate various biochemical reactions, particularly in amino acid and neurotransmitter metabolisms. Vitamin B6 is absorbed by organisms in its non-phosphorylated form and phosphorylated within cells via pyridoxal kinase (PLK) and pyridox-(am)-ine 5'-phosphate oxidase (PNPOx). The flavin mononucleotide-dependent PNPOx enzyme converts pyridoxine 5'-phosphate and pyridoxamine 5'-phosphate into PLP. PNPOx is vital for both biosynthesis and salvage pathways in organisms producing B6 vitamers. However, for those depending on vitamin B6 as a nutrient, PNPOx participates only in the salvage pathway. Transferring the PLP produced via PNPOx to client apo-enzymes is indispensable for their catalytic function, proper folding and targeting of specific organelles. PNPOx activity deficiencies due to inborn errors lead to severe neurological pathologies, particularly neonatal epileptic encephalopathy. PNPOx maintains PLP homeostasis through highly regulated mechanisms, including structural alterations throughout the catalytic cycle and allosteric PLP binding, influencing substrate transformation at the active site. Elucidation at the molecular level of the mechanisms underlying PNPOx activity deficiencies is a requirement to develop personalized approaches to treat related disorders. Finally, despite shared features, the few PNPOx enzymes molecularly and functionally studied show species-specific regulatory properties that open the possibility of targeting it in pathogenic organisms.


Assuntos
Doenças Metabólicas , Piridoxaminafosfato Oxidase , Humanos , Recém-Nascido , Oxirredutases , Fosfatos , Piridoxaminafosfato Oxidase/metabolismo , Fosfato de Piridoxal/metabolismo , Vitamina B 6/metabolismo , Piridoxina , Vitaminas
2.
Protein Sci ; 33(2): e4900, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38284493

RESUMO

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.


Assuntos
Oxirredutases , Piridoxaminafosfato Oxidase , Humanos , Sítio Alostérico , Oxirredutases/metabolismo , Fosfatos , Fosfato de Piridoxal/metabolismo , Piridoxaminafosfato Oxidase/genética , Piridoxaminafosfato Oxidase/metabolismo
3.
J Biol Chem ; 299(9): 105047, 2023 09.
Artigo em Inglês | MEDLINE | ID: mdl-37451483

RESUMO

Recently, biallelic variants in PLPBP coding for pyridoxal 5'-phosphate homeostasis protein (PLPHP) were identified as a novel cause of early-onset vitamin B6-dependent epilepsy. The molecular function and precise role of PLPHP in vitamin B6 metabolism are not well understood. To address these questions, we used PLPHP-deficient patient skin fibroblasts and HEK293 cells and YBL036C (PLPHP ortholog)-deficient yeast. We showed that independent of extracellular B6 vitamer type (pyridoxine, pyridoxamine, or pyridoxal), intracellular pyridoxal 5'-phosphate (PLP) was lower in PLPHP-deficient fibroblasts and HEK293 cells than controls. Culturing cells with pyridoxine or pyridoxamine led to the concentration-dependent accumulation of pyridoxine 5'-phosphate and pyridoxamine 5'-phosphate (PMP), respectively, suggesting insufficient pyridox(am)ine 5'-phosphate oxidase activity. Experiments utilizing 13C4-pyridoxine confirmed lower pyridox(am)ine 5'-phosphate oxidase activity and revealed increased fractional turnovers of PLP and pyridoxal, indicating increased PLP hydrolysis to pyridoxal in PLPHP-deficient cells. This effect could be partly counteracted by inactivation of pyridoxal phosphatase. PLPHP deficiency had a distinct effect on mitochondrial PLP and PMP, suggesting impaired activity of mitochondrial transaminases. Moreover, in YBL036C-deficient yeast, PLP was depleted and PMP accumulated only with carbon sources requiring mitochondrial metabolism. Lactate and pyruvate accumulation along with the decrease of tricarboxylic acid cycle intermediates downstream of α-ketoglutarate suggested impaired mitochondrial oxidative metabolism in PLPHP-deficient HEK293 cells. We hypothesize that impaired activity of mitochondrial transaminases may contribute to this depletion. Taken together, our study provides new insights into the pathomechanisms of PLPBP deficiency and reinforces the link between PLPHP function, vitamin B6 metabolism, and mitochondrial oxidative metabolism.


Assuntos
Mitocôndrias , Vitamina B 6 , Humanos , Células HEK293 , Proteínas/genética , Proteínas/metabolismo , Fosfato de Piridoxal/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transaminases/metabolismo , Vitamina B 6/metabolismo , Fibroblastos , Células Cultivadas , Piridoxaminafosfato Oxidase/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/enzimologia , Mitocôndrias/metabolismo , Oxirredução , Aminoácidos/metabolismo
4.
Hum Mol Genet ; 32(11): 1765-1771, 2023 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-36106796

RESUMO

BACKGROUND: To summarize the clinical and genetic characteristics of patients with pyridox(am)ine-5'-phosphate oxidase (PNPO) deficiency. METHODS: Clinical and genetic data of the patients were collected and analyzed. RESULTS: Eighteen patients from 17 families with variants in PNPO were collected, and 15 cases survived to date. The age of onset ranged from 1 day to 5 months (median age 6.5 days) and seven of them presented with seizures <24 h. About 7/18 (39%) of patients showed seizure-free with pyridoxine (PN) or pyridoxal-5'-phosphate treatment. Two patients showed surprised therapeutic responses to antiseizure medications therapy: one could be controlled for up to 1 year and 5 months, and the other showed seizure-free for >8 years. The neurodevelopment was normal in one patient, mild delay in four, in whom responded well to PN. Severe delay could be seen in the remaining 10 surviving patients. Genetic analysis revealed 14 variants of PNPO, seven of which were novel. Five pairs of unrelated patients were observed to carry the same variants, respectively, and had similar developmental status and onset age of seizures in some degree in each pair, whereas also had differences. CONCLUSIONS: The clinical characteristics, including age of onset, treatment response and prognosis, were variable and difficult to classify into different types clearly. Patients with PNPO deficiency who used PN as their main treatment and being able to control seizures seemed to be associated with better outcomes. Patients with the same genotype tended to show the correlation of phenotype-genotype.


Assuntos
Encefalopatias Metabólicas , Hipóxia-Isquemia Encefálica , Doenças Metabólicas , Piridoxaminafosfato Oxidase , Humanos , Encefalopatias Metabólicas/genética , Hipóxia-Isquemia Encefálica/genética , Oxirredutases , Fosfatos/uso terapêutico , Fosfato de Piridoxal/uso terapêutico , Piridoxaminafosfato Oxidase/deficiência , Piridoxaminafosfato Oxidase/genética , Piridoxina , Convulsões/tratamento farmacológico , Convulsões/genética
5.
Proc Natl Acad Sci U S A ; 119(9)2022 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-35217610

RESUMO

Pyridox(am)ine 5 ' -phosphate oxidase (PNPO) catalyzes the rate-limiting step in the synthesis of pyridoxal 5 ' -phosphate (PLP), the active form of vitamin B6 required for the synthesis of neurotransmitters gamma-aminobutyric acid (GABA) and the monoamines. Pathogenic variants in PNPO have been increasingly identified in patients with neonatal epileptic encephalopathy and early-onset epilepsy. These patients often exhibit different types of seizures and variable comorbidities. Recently, the PNPO gene has also been implicated in epilepsy in adults. It is unclear how these phenotypic variations are linked to specific PNPO alleles and to what degree diet can modify their expression. Using CRISPR-Cas9, we generated four knock-in Drosophila alleles, hWT , hR116Q , hD33V , and hR95H , in which the endogenous Drosophila PNPO was replaced by wild-type human PNPO complementary DNA (cDNA) and three epilepsy-associated variants. We found that these knock-in flies exhibited a wide range of phenotypes, including developmental impairments, abnormal locomotor activities, spontaneous seizures, and shortened life span. These phenotypes are allele dependent, varying with the known biochemical severity of these mutations and our characterized molecular defects. We also showed that diet treatments further diversified the phenotypes among alleles, and PLP supplementation at larval and adult stages prevented developmental impairments and seizures in adult flies, respectively. Furthermore, we found that hR95H had a significant dominant-negative effect, rendering heterozygous flies susceptible to seizures and premature death. Together, these results provide biological bases for the various phenotypes resulting from multifunction of PNPO, specific molecular and/or genetic properties of each PNPO variant, and differential allele-diet interactions.


Assuntos
Alelos , Dieta , Epilepsia/genética , Fenótipo , Piridoxaminafosfato Oxidase/genética , Vitamina B 6/metabolismo , Sequência de Aminoácidos , Animais , Drosophila melanogaster , Humanos , Piridoxaminafosfato Oxidase/química , Homologia de Sequência de Aminoácidos
6.
Int J Mol Sci ; 24(1)2022 Dec 30.
Artigo em Inglês | MEDLINE | ID: mdl-36614085

RESUMO

Pyridoxal 5'-phosphate (PLP), the active form of vitamin B6, serves as a cofactor for scores of B6-dependent (PLP-dependent) enzymes involved in many cellular processes. One such B6 enzyme is dopa decarboxylase (DDC), which is required for the biosynthesis of key neurotransmitters, e.g., dopamine and serotonin. PLP-dependent enzymes are biosynthesized as apo-B6 enzymes and then converted to the catalytically active holo-B6 enzymes by Schiff base formation between the aldehyde of PLP and an active site lysine of the protein. In eukaryotes, PLP is made available to the B6 enzymes through the activity of the B6-salvage enzymes, pyridoxine 5'-phosphate oxidase (PNPO) and pyridoxal kinase (PLK). To minimize toxicity, the cell keeps the content of free PLP (unbound) very low through dephosphorylation and PLP feedback inhibition of PNPO and PLK. This has led to a proposed mechanism of complex formation between the B6-salvage enzymes and apo-B6 enzymes prior to the transfer of PLP, although such complexes are yet to be characterized at the atomic level, presumably due to their transient nature. A computational study, for the first time, was used to predict a likely PNPO and DDC complex, which suggested contact between the allosteric PLP tight-binding site on PNPO and the active site of DDC. Using isothermal calorimetry and/or surface plasmon resonance, we also show that PNPO binds both apoDDC and holoDDC with dissociation constants of 0.93 ± 0.07 µM and 2.59 ± 0.11 µM, respectively. Finally, in the presence of apoDDC, the tightly bound PLP on PNPO is transferred to apoDDC, resulting in the formation of about 35% holoDDC.


Assuntos
Piridoxaminafosfato Oxidase , Piridoxina , Piridoxaminafosfato Oxidase/metabolismo , Dopa Descarboxilase , Fosfato de Piridoxal/metabolismo , Oxirredutases , Piridoxal Quinase/metabolismo
7.
Int J Mol Sci ; 22(21)2021 Nov 06.
Artigo em Inglês | MEDLINE | ID: mdl-34769443

RESUMO

Several variants of the enzyme pyridox(am)ine 5'-phosphate oxidase (PNPO), responsible for a rare form of vitamin B6-dependent neonatal epileptic encephalopathy known as PNPO deficiency (PNPOD), have been reported. However, only a few of them have been characterised with respect to their structural and functional properties, despite the fact that the knowledge of how variants affect the enzyme may clarify the disease mechanism and improve treatment. Here, we report the characterisation of the catalytic, allosteric and structural properties of recombinantly expressed D33V, R161C, P213S, and E50K variants, among which D33V (present in approximately 10% of affected patients) is one of the more common variants responsible for PNPOD. The D33V and E50K variants have only mildly altered catalytic properties. In particular, the E50K variant, given that it has been found on the same chromosome with other known pathogenic variants, may be considered non-pathogenic. The P213S variant has lower thermal stability and reduced capability to bind the FMN cofactor. The variant involving Arg161 (R161C) largely decreases the affinity for the pyridoxine 5'-phosphate substrate and completely abolishes the allosteric feedback inhibition exerted by the pyridoxal 5'-phosphate product.


Assuntos
Encefalopatias Metabólicas/genética , Epilepsia/genética , Hipóxia-Isquemia Encefálica/genética , Mutação , Fosfato de Piridoxal/análogos & derivados , Piridoxaminafosfato Oxidase/deficiência , Piridoxaminafosfato Oxidase/genética , Convulsões/genética , Vitamina B 6/metabolismo , Encefalopatias Metabólicas/metabolismo , Encefalopatias Metabólicas/patologia , Epilepsia/metabolismo , Epilepsia/patologia , Humanos , Hipóxia-Isquemia Encefálica/metabolismo , Hipóxia-Isquemia Encefálica/patologia , Recém-Nascido , Doenças Metabólicas/etiologia , Doenças Metabólicas/metabolismo , Doenças Metabólicas/patologia , Fosfato de Piridoxal/metabolismo , Piridoxaminafosfato Oxidase/metabolismo , Convulsões/metabolismo , Convulsões/patologia , Relação Estrutura-Atividade
8.
J Biol Chem ; 296: 100795, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34019876

RESUMO

Pyridoxal 5'-phosphate (PLP), the catalytically active form of vitamin B6, 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).


Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Fosfato de Piridoxal/metabolismo , Piridoxaminafosfato Oxidase/metabolismo , Sítio Alostérico , Cristalografia por Raios X , Escherichia coli/química , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/química , Humanos , Modelos Moleculares , Conformação Proteica , Piridoxaminafosfato Oxidase/química
9.
Biochimie ; 183: 18-29, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33421502

RESUMO

Pyridoxal 5'-phosphate (PLP), the active cofactor form of vitamin B6 is required by over 160 PLP-dependent (vitamin B6) enzymes serving diverse biological roles, such as carbohydrates, amino acids, hemes, and neurotransmitters metabolism. Three key enzymes, pyridoxal kinase (PL kinase), pyridoxine 5'-phosphate oxidase (PNPO), and phosphatases metabolize and supply PLP to PLP-dependent enzymes through the salvage pathway. In born errors in the salvage enzymes are known to cause inadequate levels of PLP in the cell, particularly in neuronal cells. The resulting PLP deficiency is known to cause or implicated in several pathologies, most notably seizures. One such disorder, PNPO-dependent neonatal epileptic encephalopathy (NEE) results from natural mutations in PNPO and leads to null or reduced enzymatic activity. NEE does not respond to conventional antiepileptic drugs but may respond to treatment with the B6 vitamers PLP and/or pyridoxine (PN). In born errors that lead to PLP deficiency in cells have also been reported in PL kinase, however, to date none has been associated with epilepsy or seizure. One such pathology is polyneuropathy that responds to PLP therapy. Phosphatase deficiency or hypophosphatasia disorder due to pathogenic mutations in alkaline phosphatase is known to cause seizures that respond to PN therapy. In this article, we review the biochemical features of in born errors pertaining to the salvage enzyme's deficiency that leads to NEE and other pathologies. We also present perspective on vitamin B6 treatment for these disorders, along with attempts to develop zebrafish model to study the NEE syndrome in vivo.


Assuntos
Encefalopatias Metabólicas , Hipóxia-Isquemia Encefálica , Erros Inatos do Metabolismo , Fosfotransferases (Aceptor do Grupo Álcool) , Fosfato de Piridoxal , Piridoxaminafosfato Oxidase/deficiência , Convulsões , Animais , Encefalopatias Metabólicas/genética , Encefalopatias Metabólicas/metabolismo , Humanos , Hipóxia-Isquemia Encefálica/genética , Hipóxia-Isquemia Encefálica/metabolismo , Recém-Nascido , Erros Inatos do Metabolismo/genética , Erros Inatos do Metabolismo/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Fosfato de Piridoxal/genética , Fosfato de Piridoxal/metabolismo , Piridoxaminafosfato Oxidase/genética , Piridoxaminafosfato Oxidase/metabolismo , Convulsões/genética , Convulsões/metabolismo
11.
Clin Genet ; 99(1): 99-110, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32888189

RESUMO

Pyridoxamine-5'-phosphate oxidase (PNPO) deficiency is an autosomal recessive pyridoxal 5'-phosphate (PLP)-vitamin-responsive epileptic encephalopathy. The emerging feature of PNPO deficiency is the occurrence of refractory seizures in the first year of life. Pre-maturity and fetal distress, combined with neonatal seizures, are other associated key characteristics. The phenotype results from a dependency of PLP which regulates several enzymes in the body. We present the phenotypic and genotypic spectrum of (PNPO) deficiency based on a literature review (2002-2020) of reports (n = 33) of patients with confirmed PNPO deficiency (n = 87). All patients who received PLP (n = 36) showed a clinical response, with a complete dramatic PLP response with seizure cessation observed in 61% of patients. In spite of effective seizure control with PLP, approximately 56% of patients affected with PLP-dependent epilepsy suffer developmental delay/intellectual disability. There is no diagnostic biomarker, and molecular testing required for diagnosis. However, we noted that cerebrospinal fluid (CSF) PLP was low in 81%, CSF glycine was high in 80% and urinary vanillactic acid was high in 91% of the cases. We observed only a weak correlation between the severity of PNPO protein disruption and disease outcomes, indicating the importance of other factors, including seizure onset and time of therapy initiation. We found that pre-maturity, the delay in initiation of PLP therapy and early onset of seizures correlate with a poor neurocognitive outcome. Given the amenability of PNPO to PLP therapy for seizure control, early diagnosis is essential.


Assuntos
Encefalopatias Metabólicas/genética , Epilepsia/genética , Hipóxia-Isquemia Encefálica/genética , Doenças Metabólicas/genética , Piridoxaminafosfato Oxidase/deficiência , Piridoxaminafosfato Oxidase/genética , Convulsões/genética , Encefalopatias Metabólicas/metabolismo , Encefalopatias Metabólicas/fisiopatologia , Epilepsia/fisiopatologia , Humanos , Hipóxia-Isquemia Encefálica/metabolismo , Hipóxia-Isquemia Encefálica/fisiopatologia , Doenças Metabólicas/metabolismo , Doenças Metabólicas/fisiopatologia , Mutação/genética , Fosfato de Piridoxal/genética , Fosfato de Piridoxal/metabolismo , Piridoxaminafosfato Oxidase/metabolismo , Convulsões/metabolismo , Convulsões/fisiopatologia
12.
J Struct Biol ; 212(3): 107645, 2020 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-33045383

RESUMO

Pyridoxal 5'-phosphate (PLP) is the active form of vitamin B6 and a cofactor for more than 140 enzymes. This coenzyme plays a pivotal role in catalysis of various enzymatic reactions that are critical for the survival of organisms. Entamoeba histolytica depends on the uptake of pyridoxal (PL), a B6 vitamer from the external environment which is then phosphorylated by pyridoxal kinase (EhPLK) to form PLP via the salvage pathway. E. histolytica cannot synthesise vitamin B6de-novo, and also lacks pyridoxine 5'-phosphate oxidase, a salvage pathway enzyme required to produce PLP from pyridoxine phosphate (PNP) and pyridoxamine phosphate (PMP). Analysing the importance of PLK in E. histolytica, we have determined the high-resolution crystal structures of the dimeric pyridoxal kinase in apo, ADP-bound, and PLP-bound states. These structures provided a snapshot of the transition state and help in understanding the reaction mechanism in greater detail. The EhPLK structure significantly differed from the human homologue at its PLP binding site, and the phylogenetic study also revealed its divergence from human PLK. Further, gene regulation of EhPLK using sense and antisense RNA showed that any change in optimal level is harmful to the pathogen. Biochemical and in vivo studies unveiled EhPLK to be essential for this pathogen, while the molecular differences with human PLK structure can be exploited for the structure-guided design of EhPLK inhibitors.


Assuntos
Entamoeba histolytica/metabolismo , Piridoxal Quinase/metabolismo , Sítios de Ligação/fisiologia , Catálise , Fosforilação/fisiologia , Filogenia , Fosfato de Piridoxal/análogos & derivados , Fosfato de Piridoxal/metabolismo , Piridoxamina/análogos & derivados , Piridoxamina/metabolismo , Piridoxaminafosfato Oxidase/metabolismo , Vitamina B 6/metabolismo
13.
Sci Rep ; 10(1): 13621, 2020 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-32788630

RESUMO

Defects of vitamin B6 metabolism are responsible for severe neurological disorders, such as pyridoxamine 5'-phosphate oxidase deficiency (PNPOD; OMIM: 610090), an autosomal recessive inborn error of metabolism that usually manifests with neonatal-onset severe seizures and subsequent encephalopathy. At present, 27 pathogenic mutations of the gene encoding human PNPO are known, 13 of which are homozygous missense mutations; however, only 3 of them have been characterised with respect to the molecular and functional properties of the variant enzyme forms. Moreover, studies on wild type and variant human PNPOs have so far largely ignored the regulation properties of this enzyme. Here, we present a detailed characterisation of the inhibition mechanism of PNPO by pyridoxal 5'-phosphate (PLP), the reaction product of the enzyme. Our study reveals that human PNPO has an allosteric PLP binding site that plays a crucial role in the enzyme regulation and therefore in the regulation of vitamin B6 metabolism in humans. Furthermore, we have produced, recombinantly expressed and characterised several PNPO pathogenic variants responsible for PNPOD (G118R, R141C, R225H, R116Q/R225H, and X262Q). Such replacements mainly affect the catalytic activity of PNPO and binding of the enzyme substrate and FMN cofactor, leaving the allosteric properties unaltered.


Assuntos
Encefalopatias Metabólicas/genética , Hipóxia-Isquemia Encefálica/genética , Fosfato de Piridoxal/metabolismo , Piridoxaminafosfato Oxidase/química , Piridoxaminafosfato Oxidase/deficiência , Piridoxaminafosfato Oxidase/metabolismo , Convulsões/genética , Regulação Alostérica , Sítio Alostérico , Domínio Catalítico , Cristalografia por Raios X , Mononucleotídeo de Flavina/metabolismo , Variação Genética , Humanos , Modelos Moleculares , Conformação Proteica , Piridoxaminafosfato Oxidase/genética
14.
Front Immunol ; 11: 622162, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33613557

RESUMO

Schistosomes are parasitic platyhelminths that currently infect >200 million people globally. The adult worms can live within the vasculature of their hosts for many years where they acquire all nutrients necessary for their survival and growth. In this work we focus on how Schistosoma mansoni parasites acquire and metabolize vitamin B6, whose active form is pyridoxal phosphate (PLP). We show here that live intravascular stage parasites (schistosomula and adult males and females) can cleave exogenous PLP to liberate pyridoxal. Of the three characterized nucleotide-metabolizing ectoenzymes expressed at the schistosome surface (SmAP, SmNPP5, and SmATPDase1), only SmAP hydrolyzes PLP. Heat-inactivated recombinant SmAP can no longer cleave PLP. Further, parasites whose SmAP gene has been suppressed by RNAi are significantly impaired in their ability to cleave PLP compared to controls. When schistosomes are incubated in murine plasma, they alter its metabolomic profile-the levels of both pyridoxal and phosphate increase over time, a finding consistent with the action of host-exposed SmAP acting on PLP. We hypothesize that SmAP-mediated dephosphorylation of PLP generates a pool of pyridoxal around the worms that can be conveniently taken in by the parasites to participate in essential, vitamin B6-driven metabolism. In addition, since host PLP-dependent enzymes play active roles in inflammatory processes, parasite-mediated cleavage of this metabolite may serve to limit parasite-damaging inflammation. In this work we also identified schistosome homologs of enzymes that are involved in intracellular vitamin B6 metabolism. These are pyridoxal kinase (SmPK) as well as pyridoxal phosphate phosphatase (SmPLP-Ph) and pyridox(am)ine 5'-phosphate oxidase (SmPNPO) and cDNAs encoding these three enzymes were cloned and sequenced. The three genes encoding these enzymes all display high relative expression in schistosomula and adult worms suggestive of robust vitamin B6 metabolism in the intravascular life stages.


Assuntos
Fosfatase Alcalina/metabolismo , Fosfato de Piridoxal/sangue , Schistosoma mansoni/metabolismo , Vitamina B 6/metabolismo , Fosfatase Alcalina/genética , Sequência de Aminoácidos , Animais , Feminino , Regulação da Expressão Gênica no Desenvolvimento/genética , Masculino , Camundongos , Fosfatos/sangue , Monoéster Fosfórico Hidrolases/sangue , Monoéster Fosfórico Hidrolases/genética , Fosforilação , Filogenia , Piridoxal/sangue , Piridoxal Quinase/sangue , Piridoxal Quinase/genética , Fosfato de Piridoxal/metabolismo , Piridoxaminafosfato Oxidase/sangue , Piridoxaminafosfato Oxidase/genética , Interferência de RNA , Proteínas Recombinantes , Schistosoma mansoni/enzimologia , Schistosoma mansoni/genética , Schistosoma mansoni/crescimento & desenvolvimento , Alinhamento de Sequência
15.
J Cell Physiol ; 235(1): 504-512, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31506944

RESUMO

Pyridoxine/pyridoxamine 5'-phosphate oxidase (PNPO) and pyridoxal kinase (PDXK) cooperate to produce pyridoxal 5'-phosphate (PLP), the active form of vitamin B6. PDXK phosphorylates pyridoxine, pyridoxamine, and pyridoxal by producing PNP, PMP, and PLP, whereas PNPO oxidizes PNP, PMP, into PLP. We previously demonstrated that PDXK depletion in Drosophila and human cells impacts on glucose metabolism and DNA integrity. Here we characterized sgll, the Drosophila ortholog of PNPO gene, showing that its silencing by RNA interference elicits chromosome aberrations (CABs) in brains and induces diabetic hallmarks such as hyperglycemia and small body size. We showed that in sgllRNAi neuroblasts CABs are largely produced by the genotoxic effect of the advanced glycation end products triggered by high glucose. As in sgllRNAi cells, part of PLP is still produced by PDXK activity, these data suggest that PLP dosage need to be tightly regulated to guarantee glucose homeostasis and DNA integrity.


Assuntos
Drosophila melanogaster/metabolismo , Piridoxal Quinase/metabolismo , Fosfato de Piridoxal/biossíntese , Piridoxaminafosfato Oxidase/metabolismo , Animais , Aberrações Cromossômicas , DNA/fisiologia , Glucose/metabolismo , Produtos Finais de Glicação Avançada/metabolismo , Hiperglicemia/genética , Modelos Animais , Piridoxaminafosfato Oxidase/genética , Interferência de RNA , RNA Interferente Pequeno/genética
16.
J Inherit Metab Dis ; 43(1): 2-13, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31222759

RESUMO

Inborn errors of metabolism cause disease because of accumulation of a metabolite before the blocked step or deficiency of an essential metabolite downstream of the block. Treatments can be directed at reducing the levels of a toxic metabolite or correcting a metabolite deficiency. Many disorders have been treated successfully first in a single patient because we can measure the metabolites and adjust treatment to get them as close as possible to the normal range. Examples are drawn from Komrower's description of treatment of homocystinuria and the author's trials of treatment in bile acid synthesis disorders (3ß-hydroxy-Δ5 -C27 -steroid dehydrogenase deficiency and Δ4 -3-oxosteroid 5ß-reductase deficiency), neurotransmitter amine disorders (aromatic L-amino acid decarboxylase [AADC] and tyrosine hydroxylase deficiencies), and vitamin B6 disorders (pyridox(am)ine phosphate oxidase deficiency and pyridoxine-dependent epilepsy [ALDH7A1 deficiency]). Sometimes follow-up shows there are milder and more severe forms of the disease and even variable clinical manifestations but by measuring the metabolites we can adjust the treatment to get the metabolites into the normal range. Biochemical measurements are not subject to placebo effects and will also show if the disorder is improving spontaneously. The hypothesis that can then be tested for clinical outcome is whether getting metabolite(s) into a target range leads to an improvement in an outcome parameter such as abnormal liver function tests, hypokinesia, epilepsy control etc. The metabolite-guided approach to treatment is an example of personalized medicine and is a better way of determining efficacy for disorders of variable severity than a randomized controlled clinical trial.


Assuntos
3-Hidroxiesteroide Desidrogenases/deficiência , Ácidos e Sais Biliares/sangue , Epilepsia/etiologia , Vitamina B 6/metabolismo , Administração Oral , Ácidos e Sais Biliares/biossíntese , Epilepsia/tratamento farmacológico , Epilepsia/metabolismo , Humanos , Doenças Metabólicas/tratamento farmacológico , Doenças Metabólicas/fisiopatologia , Fosfato de Piridoxal/uso terapêutico , Piridoxaminafosfato Oxidase/deficiência , Piridoxina/uso terapêutico , Ensaios Clínicos Controlados Aleatórios como Assunto , Deficiência de Vitamina B 6/complicações
17.
Biochim Biophys Acta Mol Basis Dis ; 1866(3): 165607, 2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-31759955

RESUMO

Pyridox(am)ine 5'-phosphate oxidase (PNPO) catalyzes oxidation of pyridoxine 5'-phosphate (PNP) and pyridoxamine 5'-phosphate (PMP) to pyridoxal 5'-phosphate (PLP), the active form of vitamin B6. PNPO deficiency results in neonatal/infantile seizures and neurodevelopmental delay. To gain insight into this disorder we generated Pnpo deficient (pnpo-/-) zebrafish (CRISPR/Cas9 gene editing). Locomotion analysis showed that pnpo-/- zebrafish develop seizures resulting in only 38% of pnpo-/- zebrafish surviving beyond 20 days post fertilization (dpf). The age of seizure onset varied and survival after the onset was brief. Biochemical profiling at 20 dpf revealed a reduction of PLP and pyridoxal (PL) and accumulation of PMP and pyridoxamine (PM). Amino acids involved in neurotransmission including glutamate, γ-aminobutyric acid (GABA) and glycine were decreased. Concentrations of several, mostly essential, amino acids were increased in pnpo-/- zebrafish suggesting impaired activity of PLP-dependent transaminases involved in their degradation. PLP treatment increased survival at 20 dpf and led to complete normalization of PLP, PL, glutamate, GABA and glycine. However, amino acid profiles only partially normalized and accumulation of PMP and PM persisted. Taken together, our data indicate that not only decreased PLP but also accumulation of PMP may play a role in the clinical phenotype of PNPO deficiency.


Assuntos
Encefalopatias Metabólicas/metabolismo , Hipóxia-Isquemia Encefálica/metabolismo , Doenças Metabólicas/etiologia , Doenças Metabólicas/metabolismo , Piridoxaminafosfato Oxidase/deficiência , Convulsões/etiologia , Convulsões/metabolismo , Peixe-Zebra/metabolismo , Aminoácidos/metabolismo , Animais , Encefalopatias Metabólicas/etiologia , Oxirredutases/metabolismo , Fosfato de Piridoxal/análogos & derivados , Fosfato de Piridoxal/metabolismo , Piridoxamina/metabolismo , Piridoxaminafosfato Oxidase/metabolismo , Transmissão Sináptica/fisiologia
18.
Biochem J ; 476(20): 3033-3052, 2019 10 30.
Artigo em Inglês | MEDLINE | ID: mdl-31657440

RESUMO

6-NADH and 6-NADPH are strong inhibitors of several dehydrogenases that may form spontaneously from NAD(P)H. They are known to be oxidized to NAD(P)+ by mammalian renalase, an FAD-linked enzyme mainly present in heart and kidney, and by related bacterial enzymes. We partially purified an enzyme oxidizing 6-NADPH from rat liver, and, surprisingly, identified it as pyridoxamine-phosphate oxidase (PNPO). This was confirmed by the finding that recombinant mouse PNPO oxidized 6-NADH and 6-NADPH with catalytic efficiencies comparable to those observed with pyridoxine- and pyridoxamine-5'-phosphate. PNPOs from Escherichia coli, Saccharomyces cerevisiae and Arabidopsis thaliana also displayed 6-NAD(P)H oxidase activity, indicating that this 'side-activity' is conserved. Remarkably, 'pyridoxamine-phosphate oxidase-related proteins' (PNPO-RP) from Nostoc punctiforme, A. thaliana and the yeast S. cerevisiae (Ygr017w) were not detectably active on pyridox(am)ine-5'-P, but oxidized 6-NADH, 6-NADPH and 2-NADH suggesting that this may be their main catalytic function. Their specificity profiles were therefore similar to that of renalase. Inactivation of renalase and of PNPO in mammalian cells and of Ygr017w in yeasts led to the accumulation of a reduced form of 6-NADH, tentatively identified as 4,5,6-NADH3, which can also be produced in vitro by reduction of 6-NADH by glyceraldehyde-3-phosphate dehydrogenase or glucose-6-phosphate dehydrogenase. As 4,5,6-NADH3 is not a substrate for renalase, PNPO or PNPO-RP, its accumulation presumably reflects the block in the oxidation of 6-NADH. These findings indicate that two different classes of enzymes using either FAD (renalase) or FMN (PNPOs and PNPO-RPs) as a cofactor play an as yet unsuspected role in removing damaged forms of NAD(P).


Assuntos
Biocatálise , NADPH Oxidases/metabolismo , NAD/metabolismo , Piridoxaminafosfato Oxidase/metabolismo , Animais , Arabidopsis/enzimologia , Domínio Catalítico , Escherichia coli/enzimologia , Técnicas de Inativação de Genes , Células HCT116 , Humanos , Fígado/enzimologia , Camundongos , Monoaminoxidase/química , Monoaminoxidase/metabolismo , NADPH Oxidases/isolamento & purificação , Nostoc/enzimologia , Oxirredução , Piridoxaminafosfato Oxidase/química , Ratos , Saccharomyces cerevisiae/enzimologia , Transfecção
20.
J Biol Chem ; 294(43): 15593-15603, 2019 10 25.
Artigo em Inglês | MEDLINE | ID: mdl-31484724

RESUMO

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.


Assuntos
Escherichia coli/enzimologia , Retroalimentação Fisiológica , Piridoxaminafosfato Oxidase/antagonistas & inibidores , Regulação Alostérica , Sítios de Ligação , Biocatálise , Cinética , Modelos Moleculares , Oxirredução , Fosfato de Piridoxal/análogos & derivados , Fosfato de Piridoxal/metabolismo , Piridoxaminafosfato Oxidase/química , Piridoxaminafosfato Oxidase/metabolismo , Análise Espectral
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