-
Protein Science : a Publication of the... Feb 2024Adequate levels of pyridoxal 5'-phosphate (PLP), the catalytically active form of vitamin B , and its proper distribution in the body are essential for human health. The...
Adequate levels of pyridoxal 5'-phosphate (PLP), the catalytically active form of vitamin B , 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.
Topics: Humans; Allosteric Site; Oxidoreductases; Phosphates; Pyridoxal Phosphate; Pyridoxaminephosphate Oxidase
PubMed: 38284493
DOI: 10.1002/pro.4900 -
Scientific Reports Jan 2024Limited studies are available on vitamin B6 status in domestic cats. To this end, we evaluated glutamate-oxaloacetate transaminase (GOT) activity in hemolysates with and...
Limited studies are available on vitamin B6 status in domestic cats. To this end, we evaluated glutamate-oxaloacetate transaminase (GOT) activity in hemolysates with and without pyridoxal 5'-phosphate addition in two feline populations: a cohort of 60 healthy, domestic (sexually intact and specific pathogen-free) cats maintained under strictly controlled conditions with appropriate diets housed at the Feline Nutrition and Pet Care Center, and a cohort of 57 cats randomly selected between December 2022 to January 2023 that visited the Veterinary Medicine Teaching Hospital to seek care under different circumstances. The GOT activity expressed as the ratio with and without pyridoxal 5'-phosphate addition (primary activation ratio; PAR) decreased significantly with age in the healthy cohort. The PAR values normalized to age established a cut-off for vitamin B6 deficiency in both cohorts, identifying 17 of 101 animals as vitamin B6 deficient. Using machine learning, a partition-based model (decision tree) was built to identify the most important factors that predicted vitamin B6 deficiency while using the resulting tree to make predictions for new observations. This analysis, performed with all 101 cats, revealed that the diagnosis of an infectious, chronic or acute condition (0.55) was the main contributor, followed by age (0.26), and body condition score (optimal-overweight; 0.19). Thus, our study supports that vitamin B6 supplementation may be indicated in junior to adult animals diagnosed with an infectious, chronic, or acute conditions or healthy cats with body weight ranging from optimal to overweight. In older cats, even if healthy, underweight to optimal cats appear to be at risk of vitamin B6 deficiency.
Topics: Animals; Cats; Hospitals, Teaching; Overweight; Phosphates; Pyridoxal Phosphate; Pyridoxine; Vitamin B 6; Vitamin B 6 Deficiency
PubMed: 38263201
DOI: 10.1038/s41598-024-52367-y -
International Journal of Molecular... Jan 2024The organic sulfur-containing compounds glucosinolates (GSLs) and the novel gasotransmitter HS are known to have cardioprotective effects. This study investigated the...
The organic sulfur-containing compounds glucosinolates (GSLs) and the novel gasotransmitter HS are known to have cardioprotective effects. This study investigated the antioxidant effects and HS-releasing potential of three GSLs ((3)-4-(methylsulfanyl)but-3-enyl GSL or glucoraphasatin, 4-hydroxybenzyl GSL or glucosinalbin, and ()-6-(methylsulfinyl)hexyl GSL or glucohesperin) in rat cardiac cells. It was found that all three GSLs had no effect on cardiac cell viability but were able to protect against HO-induced oxidative stress and cell death. NaHS, a HS donor, also protected the cells from HO-stimulated oxidative stress and cell death. The GSLs alone or mixed with cysteine, -acetylcysteine, glutathione, HO, iron and pyridoxal-5'-phosphate, or mouse liver lysates did not induce HS release. The addition of GSLs also did not alter endogenous HS levels in cardiac cells. HO significantly induced cysteine oxidation in the cystathionine gamma-lyase (CSE) protein and inhibited the HS production rate. In conclusion, this study found that the three tested GSLs protect cardiomyocytes from oxidative stress and cell death but independently of HS signaling.
Topics: Mice; Animals; Rats; Antioxidants; Glucosinolates; Hydrogen Peroxide; Myocytes, Cardiac; Acetylcysteine; Pyridoxal Phosphate
PubMed: 38255773
DOI: 10.3390/ijms25020696 -
Frontiers in Endocrinology 2023Hypophosphatasia (HPP) is an inborn metabolic error caused by mutations in the ALPL gene encoding tissue non-specific alkaline phosphatase (TNSALP) and leading to...
INTRODUCTION
Hypophosphatasia (HPP) is an inborn metabolic error caused by mutations in the ALPL gene encoding tissue non-specific alkaline phosphatase (TNSALP) and leading to decreased alkaline phosphatase (ALP) activity. Although the main characteristic of this disease is bone involvement, it presents a great genetic and clinical variability, which makes it a systemic disease.
METHODS
Patients were recruited based on biochemical assessments. Diagnosis was made by measuring serum ALP and pyridoxal 5-phosphate levels and finally by Sanger sequencing of the ALPL gene from peripheral blood mononuclear cells. Characterization of the new variants was performed by transfection of the variants into HEK293T cells, where ALP activity and cellular localization were measured by flow cytometry. The dominant negative effect was analyzed by co-transfection of each variant with the wild-type gene, measuring ALP activity and analyzing cellular localization by flow cytometry.
RESULTS
Two previously undescribed variants were found in the ALPL gene: leucine 6 to serine missense mutation (c.17T>C, L6S) affecting the signal peptide and threonine 167 deletion (c.498_500delCAC, T167del) affecting the vicinity of the active site. These mutations lead mainly to non-pathognomonic symptoms of HPP. Structural prediction and modeling tools indicated the affected residues as critical residues with important roles in protein structure and function. In vitro results demonstrated low TNSALP activity and a dominant negative effect in both mutations. The results of the characterization of these variants suggest that the pleiotropic role of TNSALP could be involved in the systemic effects observed in these patients highlighting digestive and autoimmune disorders associated with TNSALP dysfunction.
CONCLUSIONS
The two new mutations have been classified as pathogenic. At the clinical level, this study suggests that both mutations not only lead to pathognomonic symptoms of the disease, but may also play a role at the systemic level.
Topics: Humans; Hypophosphatasia; Alkaline Phosphatase; HEK293 Cells; Leukocytes, Mononuclear; Mutation
PubMed: 38234425
DOI: 10.3389/fendo.2023.1320516 -
Redox Biology Feb 2024Supersulfides, which are defined as sulfur species with catenated sulfur atoms, are increasingly being investigated in biology. We recently identified pyridoxal...
Supersulfides, which are defined as sulfur species with catenated sulfur atoms, are increasingly being investigated in biology. We recently identified pyridoxal phosphate (PLP)-dependent biosynthesis of cysteine persulfide (CysSSH) and related supersulfides by cysteinyl-tRNA synthetase (CARS). Here, we investigated the physiological role of CysSSH in budding yeast (Saccharomyces cerevisiae) by generating a PLP-binding site mutation K109A in CRS1 (the yeast ortholog of CARS), which decreased the synthesis of CysSSH and related supersulfides and also led to reduced chronological aging, effects that were associated with an increased endoplasmic reticulum stress response and impaired mitochondrial bioenergetics. Reduced chronological aging in the K109A mutant could be rescued by using exogenous supersulfide donors. Our findings indicate important roles for CARS in the production and metabolism of supersulfides-to mediate mitochondrial function and to regulate longevity.
Topics: Longevity; Mitochondria; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Sulfur
PubMed: 38199039
DOI: 10.1016/j.redox.2023.103018 -
Food & Nutrition Research 2023Pyridoxal 5´-phosphate (PLP) is the main form of vitamin B in animal tissue and functions as a coenzyme for more than 160 different enzymatic reactions in the... (Review)
Review
Pyridoxal 5´-phosphate (PLP) is the main form of vitamin B in animal tissue and functions as a coenzyme for more than 160 different enzymatic reactions in the metabolism of amino acids, carbohydrates, lipids, and neurotransmitters. Estimated dietary intake of vitamin B and plasma PLP values differ a lot between studies, something which may be due to variable use of supplements, variations in dietary assessment and analytical methods. These factors make it difficult to achieve precise data for setting a correct recommended intake of vitamin B. In addition, a plasma PLP concentration of 30 nmol/L is considered to be sufficient and the current recommendations for vitamin B intake is based on this concept. However, the metabolic marker for vitamin B status, HK ratio (HKr), starts to increase already when plasma PLP falls below 100 nmol/L and increases more steeply below 50 nmol/L, indicating biochemical deficiency. Consequently, a plasma PLP concentration of 30 nmol/L, may be too low as a marker for an adequate vitamin B status.
PubMed: 38187791
DOI: 10.29219/fnr.v67.10259 -
Frontiers in Aging Neuroscience 2023Alzheimer's disease is a prevalent disease with a heavy global burden and is suggested to be a metabolic disease in the brain in recent years. The metabolome is...
INTRODUCTION
Alzheimer's disease is a prevalent disease with a heavy global burden and is suggested to be a metabolic disease in the brain in recent years. The metabolome is considered to be the most promising phenotype which reflects changes in genetic, transcript, and protein profiles as well as environmental effects. Aiming to obtain a comprehensive understanding and convenient diagnosis of MCI and AD from another perspective, researchers are working on AD metabolomics. Urine is more convenient which could reflect the change of disease at an earlier stage. Thus, we conducted a cross-sectional study to investigate novel diagnostic panels.
METHODS
We first enrolled participants from China-Japan Friendship Hospital from April 2022 to November 2022, collected urine samples and conducted an LC-MS/MS analysis. In parallel, clinical data were collected and clinical examinations were performed. After statistical and bioinformatics analyzes, significant risk factors and differential urinary metabolites were determined. We attempt to investigate diagnostic panels based on machine learning including LASSO and SVM.
RESULTS
Fifty-seven AD patients, 43 MCI patients and 62 CN subjects were enrolled. A total of 2,140 metabolites were identified among which 125 significantly differed between the AD and CN groups, including 46 upregulated ones and 79 downregulated ones. In parallel, there were 93 significant differential metabolites between the MCI and CN groups, including 23 upregulated ones and 70 downregulated ones. AD diagnostic panel (30 metabolites+ age + APOE) achieved an AUC of 0.9575 in the test set while MCI diagnostic panel (45 metabolites+ age + APOE) achieved an AUC of 0.7333 in the test set. Atropine, S-Methyl-L-cysteine-S-oxide, D-Mannose 6-phosphate (M6P), Spiculisporic Acid, N-Acetyl-L-methionine, 13,14-dihydro-15-keto-tetranor Prostaglandin D2, Pyridoxal 5'-Phosphate (PLP) and 17(S)-HpDHA were considered valuable for both AD and MCI diagnosis and defined as hub metabolites. Besides, diagnostic metabolites were weakly correlated with cognitive functions.
DISCUSSION
In conclusion, the procedure is convenient, non-invasive, and useful for diagnosis, which could assist physicians in differentiating AD and MCI from CN. Atropine, M6P and PLP were evidence-based hub metabolites in AD.
PubMed: 38187356
DOI: 10.3389/fnagi.2023.1273807 -
BioRxiv : the Preprint Server For... Dec 2023Amino acids (AAs) are modular and modifiable building blocks which nature uses to synthesize both macromolecules, such as proteins, and small molecule natural products,...
Amino acids (AAs) are modular and modifiable building blocks which nature uses to synthesize both macromolecules, such as proteins, and small molecule natural products, such as alkaloids and non-ribosomal peptides (NRPs). While the 20 main proteinogenic AAs display relatively limited side-chain diversity, a wide range of non-canonical amino acids (ncAAs) exist that are not used by the ribosome for protein synthesis but contain a broad array of structural features and functional groups not found in proteinogenic AAs. In this communication, we report the discovery of the biosynthetic pathway for a new ncAA, pazamine, which contains a cyclopropane ring formed in two steps. In the first step, a chlorine is added onto the C position of lysine by a radical halogenase PazA. The cyclopropane ring is then formed in the next step by a pyridoxal-5'-phosphate-dependent enzyme, PazB, via an S2-like attack onto C to eliminate chloride. Genetic studies of this pathway in the native host, , show that pazamine and its succinylated derivative, pazamide, potentially inhibit ethylene biosynthesis in growing plants based on alterations in the root phenotype of seedlings. We further show that PazB can be utilized to make an alternative cyclobutane-containing AA. These discoveries may lead to advances in biocatalytic production of specialty chemicals and agricultural biotechnology.
PubMed: 38168212
DOI: 10.1101/2023.12.13.571568 -
Biochimica Et Biophysica Acta. Proteins... May 2024Methionine gamma lyase (MGL) is a bacterial and plant enzyme that catalyzes the conversion of methionine in methanthiol, 2-oxobutanoate and ammonia. The enzyme belongs... (Review)
Review
Methionine gamma lyase (MGL) is a bacterial and plant enzyme that catalyzes the conversion of methionine in methanthiol, 2-oxobutanoate and ammonia. The enzyme belongs to fold type I of the pyridoxal 5'-dependent family. The catalytic mechanism and the structure of wild type MGL and variants were determined in the presence of the natural substrate as well as of many sulfur-containing derivatives. Structure-function relationship studies were pivotal for MGL exploitation in the treatment of cancer, bacterial infections, and other diseases. MGL administration to cancer cells leads to methionine starvation, thus decreasing cells viability and increasing their vulnerability towards other drugs. In antibiotic therapy, MGL acts by transforming prodrugs in powerful drugs. Numerous strategies have been pursued for the delivering of MGL in vivo to prolong its bioavailability and decrease its immunogenicity. These include conjugation with polyethylene glycol and encapsulation in synthetic or natural vesicles, eventually decorated with tumor targeting molecules, such as the natural phytoestrogens daidzein and genistein. The scientific achievements in studying MGL structure, function and perspective therapeutic applications came from the efforts of many talented scientists, among which late Tatyana Demidkina to whom we dedicate this review.
Topics: Structure-Activity Relationship; Methionine; Racemethionine; Catalysis; Cell Survival
PubMed: 38147934
DOI: 10.1016/j.bbapap.2023.140991 -
International Journal of Molecular... Dec 2023The mitochondrial matrix peptidase CLPP is crucial during cell stress. Its loss causes Perrault syndrome type 3 (PRLTS3) with infertility, neurodegeneration, and a...
The mitochondrial matrix peptidase CLPP is crucial during cell stress. Its loss causes Perrault syndrome type 3 (PRLTS3) with infertility, neurodegeneration, and a growth deficit. Its target proteins are disaggregated by CLPX, which also regulates heme biosynthesis via unfolding ALAS enzymes, providing access for pyridoxal-5'-phosphate (PLP). Despite efforts in diverse organisms with multiple techniques, CLPXP substrates remain controversial. Here, avoiding recombinant overexpression, we employed complexomics in mitochondria from three mouse tissues to identify endogenous targets. A CLPP absence caused the accumulation and dispersion of CLPX-VWA8 as AAA+ unfoldases, and of PLPBP. Similar changes and CLPX-VWA8 co-migration were evident for mitoribosomal central protuberance clusters, translation factors like GFM1-HARS2, the RNA granule components LRPPRC-SLIRP, and enzymes OAT-ALDH18A1. Mitochondrially translated proteins in testes showed reductions to <30% for MTCO1-3, the mis-assembly of the complex IV supercomplex, and accumulated metal-binding assembly factors COX15-SFXN4. Indeed, heavy metal levels were increased for iron, molybdenum, cobalt, and manganese. RT-qPCR showed compensatory downregulation only for mRNA; most accumulated proteins appeared transcriptionally upregulated. Immunoblots validated VWA8, MRPL38, MRPL18, GFM1, and OAT accumulation. Co-immunoprecipitation confirmed CLPX binding to MRPL38, GFM1, and OAT, so excess CLPX and PLP may affect their activity. Our data mechanistically elucidate the mitochondrial translation fidelity deficits which underlie progressive hearing impairment in PRLTS3.
Topics: Animals; Mice; Adenosine Triphosphatases; ATPases Associated with Diverse Cellular Activities; Endopeptidase Clp; Hearing Loss; Mitochondria; Molecular Chaperones; Respiration; Protein Biosynthesis
PubMed: 38139332
DOI: 10.3390/ijms242417503