RESUMEN
OBJECTIVES: To assess the pyridoxal 5'-phosphate (PLP) content and stability of extemporaneous PLP liquids prepared from dietary supplements used for the treatment of vitamin B6 -dependent epilepsy. METHODS: Pyridoxal 5'-phosphate liquids were prepared in accordance with the guidelines given to patients from marketed 50 mg PLP dietary capsules and tablets. The PLP content and its stability were evaluated under conditions resembling the clinical setting using reverse phase HPLC and mass spectrometry. KEY FINDINGS: Pyridoxal 5'-phosphate content in most of the extemporaneously prepared liquids from dietary supplements was found to be different from the expected amount (~16-60 mg). Most of these PLP extemporaneous liquids were stable at room temperature (protected from light) after 24 h but unstable after 4 h when exposed to light. A key photodegradation product of PLP in water was confirmed as 4-pyridoxic acid 5'-phosphate (PAP). CONCLUSION: Pyridoxal 5'-phosphate tablets from Solgar® were found to be the most reliable product for the preparation of extemporaneous PLP liquids. This work highlighted the difference between the marketed PLP dietary supplements quality and the importance of proper storage of aqueous PLP. There is a need to develop pharmaceutical forms of PLP that ensure dose accuracy and avoid potentially unsafe impurities with the aim of enhancing safety and compliance.
Asunto(s)
Epilepsia , Fosfato de Piridoxal/química , Fosfato de Piridoxal/normas , Control de Calidad , Complejo Vitamínico B/química , Complejo Vitamínico B/normas , Suplementos Dietéticos/normas , Formas de Dosificación , Estabilidad de Medicamentos , Almacenaje de Medicamentos/normas , Epilepsia/tratamiento farmacológico , Soluciones Farmacéuticas , Fotólisis , Fosfato de Piridoxal/uso terapéutico , Complejo Vitamínico B/uso terapéuticoRESUMEN
The active form of vitamin B6, pyridoxal 5'-phosphate (PLP), serves as a co-factor in more than 150 enzymatic reactions. Plasma PLP has consistently been shown to be low in inflammatory conditions; there is a parallel reduction in liver PLP, but minor changes in erythrocyte and muscle PLP and in functional vitamin B6 biomarkers. Plasma PLP also predicts the risk of chronic diseases like cardiovascular disease and some cancers, and is inversely associated with numerous inflammatory markers in clinical and population-based studies. Vitamin B6 intake and supplementation improve some immune functions in vitamin B6-deficient humans and experimental animals. A possible mechanism involved is mobilization of vitamin B6 to the sites of inflammation where it may serve as a co-factor in pathways producing metabolites with immunomodulating effects. Relevant vitamin B6-dependent inflammatory pathways include vitamin B6 catabolism, the kynurenine pathway, sphingosine 1-phosphate metabolism, the transsulfuration pathway, and serine and glycine metabolism.
Asunto(s)
Inflamación/tratamiento farmacológico , Vitamina B 6/uso terapéutico , Animales , Biomarcadores/sangre , Humanos , Inmunidad/efectos de los fármacos , Inflamación/inmunología , Fosfato de Piridoxal/química , Fosfato de Piridoxal/metabolismo , Transducción de Señal , Vitamina B 6/químicaRESUMEN
Lysine decarboxylase (LDC) is a crucial enzyme for acid stress resistance and is also utilized for the biosynthesis of cadaverine, a promising building block for bio-based polyamides. We determined the crystal structure of LDC from Selenomonas ruminantium (SrLDC). SrLDC functions as a dimer and each monomer consists of two distinct domains; a PLP-binding barrel domain and a sheet domain. We also determined the structure of SrLDC in complex with PLP and cadaverine and elucidated the binding mode of cofactor and substrate. Interestingly, compared with the apo-form of SrLDC, the SrLDC in complex with PLP and cadaverine showed a remarkable structural change at the PLP binding site. The PLP binding site of SrLDC contains the highly flexible loops with high b-factors and showed an open-closed conformational change upon the binding of PLP. In fact, SrLDC showed no LDC activity without PLP supplement, and we suggest that highly flexible PLP binding site results in low PLP affinity of SrLDC. In addition, other structurally homologous enzymes also contain the flexible PLP binding site, which indicates that high flexibility at the PLP binding site and low PLP affinity seems to be a common feature of these enzyme family.
Asunto(s)
Carboxiliasas/química , Modelos Moleculares , Conformación Molecular , Fosfato de Piridoxal/química , Selenomonas/enzimología , Secuencia de Aminoácidos , Sitios de Unión , Carboxiliasas/metabolismo , Dominio Catalítico , Activación Enzimática , Unión Proteica , Fosfato de Piridoxal/metabolismo , Proteínas Recombinantes , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
Nausea and vomiting of pregnancy (NVP) remains difficult to treat. Last century, thalidomide was used to alleviate NVP, but it caused teratogenesis by interfering with angiogenesis. The gasotransmitters hydrogen sulfide (H2S) and nitric oxide are mutually dependent on each other for their angiogenesis-related functions. Pregnancy-related requirements for increased endogenous H2S could create a temporary excess of sulfite, an H2S catabolite, which is toxic and can induce nausea. Sulfite oxidase, a molybdenum-containing enzyme, catalyzes oxidation of sulfite to sulfate, which can then be excreted or reused by the body. Supplementation with molybdenum should facilitate enhanced sulfite oxidase activity, thus lowering gestationally-elevated sulfite levels in the gastrointestinal tract and easing NVP.
Asunto(s)
Molibdeno/uso terapéutico , Náuseas Matinales/terapia , Náusea/terapia , Sulfitos/efectos adversos , Catálisis , Femenino , Humanos , Sulfuro de Hidrógeno/química , Modelos Teóricos , Neovascularización Patológica , Óxido Nítrico/química , Oxígeno/química , Embarazo , Fosfato de Piridoxal/química , Sulfitos/química , OligoelementosRESUMEN
Reactive oxygen species (ROS) and reactive nitrogen species (RNS) participate in pathological tissue damage. Mitochondrial manganese superoxide dismutase (MnSOD) normally keeps ROS and RNS in check. During development of mangafodipir (MnDPDP) as a magnetic resonance imaging (MRI) contrast agent, it was discovered that MnDPDP and its metabolite manganese pyridoxyl ethyldiamine (MnPLED) possessed SOD mimetic activity. MnDPDP has been tested as a chemotherapy adjunct in cancer patients and as an adjunct to percutaneous coronary intervention in patients with myocardial infarctions, with promising results. Whereas MRI contrast depends on release of Mn(2+), the SOD mimetic activity depends on Mn(2+) that remains bound to DPDP or PLED. Calmangafodipir [Ca4Mn(DPDP)5] is stabilized with respect to Mn(2+) and has superior therapeutic activity. Ca4Mn(DPDP)5 is presently being explored as a chemotherapy adjunct in a clinical multicenter Phase II study in patients with metastatic colorectal cancer.
Asunto(s)
Antineoplásicos/uso terapéutico , Antioxidantes/uso terapéutico , Mimetismo Biológico , Ácido Edético/análogos & derivados , Etilenodiaminas/uso terapéutico , Manganeso/metabolismo , Fosfato de Piridoxal/análogos & derivados , Superóxido Dismutasa/metabolismo , Animales , Antineoplásicos/química , Antioxidantes/química , Antioxidantes/metabolismo , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/patología , Ácido Edético/química , Ácido Edético/metabolismo , Ácido Edético/uso terapéutico , Etilenodiaminas/química , Etilenodiaminas/metabolismo , Humanos , Manganeso/química , Estructura Molecular , Infarto del Miocardio/terapia , Estrés Oxidativo/efectos de los fármacos , Intervención Coronaria Percutánea , Fosfato de Piridoxal/química , Fosfato de Piridoxal/metabolismo , Fosfato de Piridoxal/uso terapéutico , Especies Reactivas de Oxígeno/metabolismo , Relación Estructura-Actividad , Resultado del TratamientoAsunto(s)
Antineoplásicos/uso terapéutico , Neoplasias/tratamiento farmacológico , Animales , Antineoplásicos/efectos adversos , Medios de Contraste/química , Medios de Contraste/uso terapéutico , Ácido Edético/análogos & derivados , Ácido Edético/química , Ácido Edético/uso terapéutico , Humanos , Fosfato de Piridoxal/análogos & derivados , Fosfato de Piridoxal/química , Fosfato de Piridoxal/uso terapéutico , Calidad de VidaRESUMEN
Human cystathionine ß-synthase (CBS) is a unique pyridoxal 5'-phosphate (PLP)-dependent enzyme that has a regulatory heme cofactor. Previous studies have demonstrated the importance of Arg-266, a residue at the heme pocket end of α-helix 8, for communication between the heme and PLP sites. In this study, we have examined the role of the conserved Thr-257 and Thr-260 residues, located at the other end of α-helix 8 on the heme electronic environment and on activity. The mutations at the two positions destabilize PLP binding, leading to lower PLP content and ~2- to ~500-fold lower activity compared with the wild-type enzyme. Activity is unresponsive to PLP supplementation, consistent with the pyridoxine-nonresponsive phenotype of the T257M mutation in a homocystinuric patient. The H(2)S-producing activities, also impacted by the mutations, show a different pattern of inhibition compared with the canonical transsulfuration reaction. Interestingly, the mutants exhibit contrasting sensitivities to the allosteric effector, S-adenosylmethionine (AdoMet); whereas T257M and T257I are inhibited, the other mutants are hyperactivated by AdoMet. All mutants showed an increased propensity of the ferrous heme to form an inactive species with a 424 nm Soret peak and exhibited significantly reduced enzyme activity in the ferrous and ferrous-CO states. Our results provide the first evidence for bidirectional transmission of information between the cofactor binding sites, suggest the additional involvement of this region in allosteric communication with the regulatory AdoMet-binding domain, and reveal the potential for independent modulation of the canonical transsulfuration versus H(2)S-generating reactions catalyzed by CBS.
Asunto(s)
Cistationina betasintasa/metabolismo , Hemo/metabolismo , Peróxido de Hidrógeno/metabolismo , Fosfato de Piridoxal/metabolismo , Regulación Alostérica , Sitios de Unión/genética , Cistationina betasintasa/química , Cistationina betasintasa/genética , Hemo/química , Homocistinuria/enzimología , Homocistinuria/genética , Homocistinuria/metabolismo , Humanos , Hierro/química , Hierro/metabolismo , Isomerismo , Cinética , Mutación , Oxidación-Reducción , Carbonilación Proteica , Fosfato de Piridoxal/química , S-Adenosilmetionina/química , S-Adenosilmetionina/metabolismo , Espectrometría de Fluorescencia , Treonina/química , Treonina/genética , Treonina/metabolismoRESUMEN
Like an alpinist continuously seeking virgin peaks to climb, Kenji Soda has investigated a variety of unique enzymes for which there was little or no information available; and by doing so he opened up a variety of new fields in enzyme science and technology. In particular, he has promoted the study of enzymes requiring vitamin B-derived cofactors such as FAD, NAD(P) and pyridoxal 5'-phosphate, shedding light on their reaction mechanisms, enzymological properties, crystal structures and potential practical applications. Highlighted in this review are the studies of enzymes acting on d-amino acids and sulphur/selenium-containing amino acids and those from thermophilic and psychrophilic bacteria.
Asunto(s)
Enzimas , Montañismo , Aminoácidos/química , Aminoácidos/metabolismo , Bacterias/enzimología , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Enzimas/química , Enzimas/metabolismo , Flavina-Adenina Dinucleótido/química , Flavina-Adenina Dinucleótido/metabolismo , Humanos , Estructura Molecular , NADP/química , NADP/metabolismo , Fosfato de Piridoxal/química , Fosfato de Piridoxal/metabolismo , Selenio/química , Selenio/metabolismo , Azufre/química , Azufre/metabolismoRESUMEN
BACKGROUND: There are three pathways of L-threonine catabolism. The enzyme L-threonine aldolase (TA) has been shown to catalyse the conversion of L-threonine to yield glycine and acetaldehyde in bacteria, fungi and plants. Low levels of TA enzymatic activity have been found in vertebrates. It has been suggested that any detectable activity is due to serine hydroxymethyltransferase and that mammals lack a genuine threonine aldolase. RESULTS: The 7-exon murine L-threonine aldolase gene (GLY1) is located on chromosome 11, spanning 5.6 kb. The cDNA encodes a 400-residue protein. The protein has 81% similarity with the bacterium Thermotoga maritima TA. Almost all known functional residues are conserved between the two proteins including Lys242 that forms a Schiff-base with the cofactor, pyridoxal-5'-phosphate. The human TA gene is located at 17q25. It contains two single nucleotide deletions, in exons 4 and 7, which cause frame-shifts and a premature in-frame stop codon towards the carboxy-terminal. Expression of human TA mRNA was undetectable by RT-PCR. In mice, TA mRNA was found at low levels in a range of adult tissues, being highest in prostate, heart and liver. In contrast, serine/threonine dehydratase, another enzyme that catabolises L-threonine, is expressed very highly only in the liver. Serine dehydratase-like 1, also was most abundant in the liver. In whole mouse embryos TA mRNA expression was low prior to E-15 increasing more than four-fold by E-17. CONCLUSION: Mice, the western-clawed frog and the zebrafish have transcribed threonine aldolase/GLY1 genes, but the human homolog is a non-transcribed pseudogene. Serine dehydratase-like 1 is a putative L-threonine catabolising enzyme.
Asunto(s)
Glicina Hidroximetiltransferasa/genética , Transcripción Genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Mapeo Cromosómico , Clonación Molecular , Codón de Terminación , Biología Computacional , Cristalografía por Rayos X , ADN Complementario/metabolismo , Embrión de Mamíferos/metabolismo , Exones , Eliminación de Gen , Regulación del Desarrollo de la Expresión Génica , Glicina Hidroximetiltransferasa/metabolismo , Humanos , Hígado/metabolismo , Lisina/química , Ratones , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Estructura Terciaria de Proteína , Seudogenes , Fosfato de Piridoxal/química , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Factores de Tiempo , Distribución TisularRESUMEN
OBJECTIVE: To determine the biochemical status of thiamin, riboflavin and pyridoxine in parturient mothers and their newborn infants in a Mediterranean region. DESIGN: Transveral study. SETTING: St Joan University Hospital and Faculty of Medicine & Health Sciences, Universitat Rovira i Virgili, Reus, Spain. SUBJECTS: 131 healthy parturient mothers, with normal pregnancies and deliveries in St Joan University Hospital, and their newborn infants. INTERVENTIONS: Erythrocyte haemolysates were prepared from maternal blood at delivery and infants' umbilical cord blood and used to measure micronutrient status using the transketolase, glutathione reductase and aspartate aminotransferase coenzyme stimulation tests. RESULTS: Maternal and infant coenzyme activities were significantly correlated, but infant coenzyme status was better than maternal, with significantly higher basal and stimulated activity (P < 0.001) and significantly lower activation coefficients (P < 0.001). Inadequate thiamin, riboflavin or pyridoxine status occured in 38.2 62.6% (50-82) of the mothers and 3.1-37.4% (4 49) of the infants; 85.2% (46/54), 12.9% (4/31) and 24.1% (12/54) of infants born to mothers with biochemical deficiency of either thiamin, riboflavin or pyridoxine, respectively also had inadequate status. Maternal deficiencies in more than one vitamin further increased the risk of infant thiamin and pyridoxine deficiency. Maternal and infant riboflavin status were significantly correlated with fetal development (e.g. length at birth, P < 0.001). The incidence of thiamin deficiency in paturient mothers in Spain was the highest out of a 12-country comparison. CONCLUSIONS: Inadequate status for each vitamin was evident in mothers and infants. Maternal status of each individual vitamin, but especially riboflavin, was affected by maternal status of the other vitamins. Infant thiamin status was the most adversely affected by maternal deficiencies in more than one vitamin. Infant riboflavin status, however, was apparently protected from adverse maternal status.
Asunto(s)
Recién Nacido/fisiología , Estado Nutricional , Periodo Posparto/fisiología , Piridoxina/sangre , Riboflavina/sangre , Tiamina/sangre , Adolescente , Adulto , Aspartato Aminotransferasas/sangre , Femenino , Sangre Fetal/enzimología , Flavina-Adenina Dinucleótido/química , Glutatión Reductasa/sangre , Hemoglobina Glucada/análisis , Humanos , Fenómenos Fisiológicos Nutricionales del Lactante , Recién Nacido/sangre , Masculino , Periodo Posparto/sangre , Embarazo , Fosfato de Piridoxal/química , España , Estadísticas no Paramétricas , Tiamina Pirofosfato/química , Transcetolasa/sangreRESUMEN
5-Aminolevulinate synthase catalyzes the first step of the heme biosynthetic pathway in nonplant higher eukaryotes. The enzyme functions as a homodimer and requires pyridoxal 5'-phosphate as its cofactor. Lysine-313 in murine erythroid aminolevulinate synthase has been identified as the residue involved in the Schiff base linkage with pyridoxal 5'-phosphate [Ferreira, G. C., Neame, P. J., & Dailey, H. A. (1993) Protein Sci. 2, 1959-1965]. However, other residues involved in binding and orienting the cofactor remain unknown. We studied the informational content of each residue within an 11 amino acid glycine-rich region, which we propose to be part of the phosphate-binding motif, based on amino acid sequence comparison with other pyridoxal 5'-phosphate-dependent enzymes and nucleotide-binding proteins. Partial random mutagenesis of this region in murine erythroid aminolevulinate synthase gene was followed by an efficient biological selection, using a hemA- Escherichia coli strain to recover functional unnatural enzymes. Among the total of 5444 variants produced, 283 were found to be functional. DNA sequencing results of 226 functional mutants indicated that most residues in this region contained a low informational content, being able to tolerate several other amino acid substitutions. However, three residues, namely, Arg-149, Gly-142, and Gly-144, were found to contain high informational content; Arg-149 was conserved in all of the functional mutants sequenced, while Gly-142 and Gly-144 could only tolerate alanine replacement. Two codon-specific random libraries of Arg-149, and Gly-142 and -144, respectively, were constructed to test further the stringency of these three positions.(ABSTRACT TRUNCATED AT 250 WORDS)