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The FEBS Journal Nov 2018Many biological functions played by current proteins were not created by evolution from scratch, rather they were obtained combining already available protein scaffolds.... (Review)
Review
Many biological functions played by current proteins were not created by evolution from scratch, rather they were obtained combining already available protein scaffolds. This is the case of MocR-like bacterial transcription factors (MocR-TFs), a subclass of GntR transcription regulators, whose structure is the outcome of the fusion between DNA-binding proteins and pyridoxal 5'-phosphate (PLP)-dependent enzymes. The resultant chimeras can count on the properties of both protein classes, i.e. the capability to recognize specific DNA sequences and to bind PLP and amino-compounds; it is the modulation of such binding properties to confer to MocR-TFs chimeras the ability to interact with effector molecules and DNA so as to regulate transcription. MocR-TFs control different metabolic processes involving vitamin B and amino acids, which are canonical ligands of PLP-dependent enzymes. However, MocR-TFs are also implicated in the metabolism of compounds that are not substrates of PLP-dependent enzymes, such as rhizopine and ectoine. Genomic analyses show that MocR-TFs are widespread among eubacteria, implying an essential role in their metabolism and highlighting the scarcity of our knowledge on these important players in microbial metabolism. Although MocR-TFs have been discovered 15 years ago, the research activity on these transcriptional regulators has only recently intensified, producing a wealth of information that needs to be brought back to general principles. This is the main task of this review, which reports and analyses the available information concerning MocR-TFs functional role, structural features, interaction with effector molecules and the characteristics of DNA transcriptional factor-binding sites of MocR-based regulatory systems.
Topics: Bacteria; Bacterial Proteins; Gene Expression Regulation, Bacterial; Pyridoxal Phosphate; Regulon; Transcription Factors
PubMed: 29974999
DOI: 10.1111/febs.14599 -
Infection, Genetics and Evolution :... Apr 2022Pulmonary tuberculosis (TB) is a serious disease burden worldwide, and its effective early diagnosis is still facing challenges. Knowledge, acquired from multi-omics...
BACKGROUND
Pulmonary tuberculosis (TB) is a serious disease burden worldwide, and its effective early diagnosis is still facing challenges. Knowledge, acquired from multi-omics integration analysis about the association between different types of differentially expressed molecules in the plasma of TB patients and the disease traits, is anticipated to improve the accuracy of TB diagnosis through the "integrative pattern".
METHODS
In this study, the lncRNA-miRNA-mRNA interaction network was constructed based on the competing endogenous RNA (ceRNA) hypothesis by integrating our previous data sets of lncRNA, mRNA, miRNA, and metabolites. Moreover, the key regulatory axis was established by co-expression analysis and verified at the level of metabolites.
RESULTS
A ceRNA regulatory network consisting of 23 lncRNAs, 10 miRNAs, and 113 mRNAs was constructed. The analysis results suggested that lncRNA (OSBPL10-AS1), miRNA (has-miR-485-5p), and mRNA (SLC23A2) might be involved in the regulation of vitamin metabolism in patients with TB. Metabolite analysis showed that compared with the normal control group, TB patients had abnormal vitamin metabolism, and the expression levels of pyridoxal phosphate, pyridoxamine phosphate, and folic acid were significantly different between the two groups (p < 0.05).
CONCLUSION
Integrated multi-omics analysis showed that vitamin metabolism disorder may be one of the pathological characteristic of TB. OSBPL10-AS1, hsa-miR-485-5p, SLC23A2, pyridoxal phosphate, pyridoxamine phosphate, and folic acid may collectively constitute the "integrative pattern" of multiple biomarkers, which may provide an accurate diagnosis of TB.
Topics: Biomarkers; Folic Acid; Gene Regulatory Networks; Humans; MicroRNAs; Pyridoxal Phosphate; Pyridoxamine; RNA, Long Noncoding; RNA, Messenger; Tuberculosis, Pulmonary; Vitamins
PubMed: 35150890
DOI: 10.1016/j.meegid.2022.105240 -
The Journal of Biological Chemistry Sep 2023Recently, biallelic variants in PLPBP coding for pyridoxal 5'-phosphate homeostasis protein (PLPHP) were identified as a novel cause of early-onset vitamin B-dependent...
Recently, biallelic variants in PLPBP coding for pyridoxal 5'-phosphate homeostasis protein (PLPHP) were identified as a novel cause of early-onset vitamin B-dependent epilepsy. The molecular function and precise role of PLPHP in vitamin B 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 B 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 C-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 B metabolism, and mitochondrial oxidative metabolism.
Topics: Humans; HEK293 Cells; Proteins; Pyridoxal Phosphate; Saccharomyces cerevisiae; Transaminases; Vitamin B 6; Fibroblasts; Cells, Cultured; Pyridoxaminephosphate Oxidase; Mitochondria; Oxidation-Reduction; Amino Acids
PubMed: 37451483
DOI: 10.1016/j.jbc.2023.105047 -
Bioscience, Biotechnology, and... Aug 2022Enzymology, the study of enzyme structures and reaction mechanisms can be considered a classical discipline. However, enzymes cannot be freely designed to catalyze... (Review)
Review
Enzymology, the study of enzyme structures and reaction mechanisms can be considered a classical discipline. However, enzymes cannot be freely designed to catalyze desired reactions yet, and enzymology is by no means a complete science. I have long studied the reaction mechanisms of enzymes related to amino acid metabolism, such as aminotransferases and racemases, which depend on pyridoxal 5'-phosphate, a coenzyme form of vitamin B6. During these studies, I have often been reminded that enzymatic reactions are extremely sophisticated processes based on chemical principles and enzyme structures, and have often been amazed at the evolutionary mechanisms that bestowed them with such structures. In this review, I described the reaction mechanism of various pyridoxal enzymes especially related to d-amino acids metabolism, whose roles in mammals have recently attracted attention. I hope to convey some of the significance and interest in enzymology through this review.
Topics: Amino Acids; Animals; Mammals; Pyridoxal Phosphate; Racemases and Epimerases; Transaminases; Vitamin B 6
PubMed: 35751623
DOI: 10.1093/bbb/zbac102 -
Molecular Genetics and Metabolism Nov 20195-Aminolevulinate (ALA) synthase (ALAS), a homodimeric pyridoxal-5'-phosphate (PLP)-dependent enzyme, catalyzes the first step of heme biosynthesis in metazoa, fungi and... (Review)
Review
5-Aminolevulinate (ALA) synthase (ALAS), a homodimeric pyridoxal-5'-phosphate (PLP)-dependent enzyme, catalyzes the first step of heme biosynthesis in metazoa, fungi and α-proteobacteria. In this review, we focus on the advances made in unraveling the mechanism of the ALAS-catalyzed reaction during the past decade. The interplay between the PLP cofactor and the protein moiety determines and modulates the multi-intermediate reaction cycle of ALAS, which involves the decarboxylative condensation of two substrates, glycine and succinyl-CoA. Substrate binding and catalysis are rapid, and product (ALA) release dominates the overall ALAS kinetic mechanism. Interconversion between a catalytically incompetent, open conformation and a catalytically competent, closed conformation is linked to ALAS catalysis. Reversion to the open conformation, coincident with ALA dissociation, defines the slowest step of the reaction cycle. These findings were further substantiated by introducing seven mutations in the16-amino acid loop that gates the active site, yielding an ALAS variant with a greatly increased rate of catalytic turnover and heightened specificity constants for both substrates. Recently, molecular dynamics (MD) simulation analysis of various dimeric ALAS forms revealed that the seven active site loop mutations caused the proteins to adopt different conformations. In particular, the emergence of a β-strand in the mutated loop, which interacted with two preexisting β-strands to form an anti-parallel three-stranded β-sheet, conferred the murine heptavariant with a more stable open conformation and prompted faster product release than wild-type mALAS2. Moreover, the dynamics of the mALAS2 active site loop anti-correlated with that of the 35 amino acid C-terminal sequence. This led us to propose that this C-terminal extension, which is absent in prokaryotic ALASs, finely tunes mammalian ALAS activity. Based on the above results, we extend our previous proposal to include that discovery of a ligand inducing the mammalian C-terminal extension to fold offers a good prospect for the development of a new drug for X-linked protoporphyria and/or other porphyrias associated with enhanced ALAS activity and/or porphyrin accumulation.
Topics: 5-Aminolevulinate Synthetase; Biosynthetic Pathways; Catalysis; Heme; Humans; Kinetics; Molecular Dynamics Simulation; Protein Conformation; Pyridoxal Phosphate; Substrate Specificity
PubMed: 31345668
DOI: 10.1016/j.ymgme.2019.06.003 -
Archives de Pediatrie : Organe Officiel... May 2017The laboratory diagnosis of hypophosphatasia (HPP) is primarily based on the precise analysis of circulating serum alkaline phosphatase (ALP) activity, determined by...
The laboratory diagnosis of hypophosphatasia (HPP) is primarily based on the precise analysis of circulating serum alkaline phosphatase (ALP) activity, determined by biochemical assays. This analysis requires specific conditions of implementation and interpretation and should always be viewed in the light of the clinical and radiological data. Concerns regarding the normal ranges of ALP with respect to age, regarding ALP values that may overlap those of normal subjects in HPP patients, regarding apparently normal ALP values in cases of proven HPP, regarding differential diagnoses that may be responsible for low ALP values outside of HPP will be discussed. High levels of pyridoxal phosphate, a substrate of APL, are of supportive value in the diagnosis of HPP.
Topics: Alkaline Phosphatase; Biomarkers; Clinical Laboratory Techniques; Diagnosis, Differential; Humans; Hypophosphatasia; Predictive Value of Tests; Pyridoxal Phosphate; Reference Values; Sensitivity and Specificity; Vitamin B Complex
PubMed: 29405933
DOI: 10.1016/S0929-693X(18)30015-0 -
Journal of Inherited Metabolic Disease Sep 2023Over the past two decades, the field of vitamin B -dependent epilepsies has evolved by the recognition of a growing number of gene defects (ALDH7A1, PNPO, ALPL, ALDH4A1,... (Review)
Review
Over the past two decades, the field of vitamin B -dependent epilepsies has evolved by the recognition of a growing number of gene defects (ALDH7A1, PNPO, ALPL, ALDH4A1, PLPBP as well as defects of the glycosylphosphatidylinositol anchor proteins) that all lead to reduced availability of pyridoxal 5'-phosphate, an important cofactor in neurotransmitter and amino acid metabolism. In addition, positive pyridoxine response has been observed in other monogenic defects such as MOCS2 deficiency or KCNQ2 and there may be more defects to be discovered. Most entities lead to neonatal onset pharmaco-resistant myoclonic seizures or even status epilepticus and pose an emergency to the treating physician. Research has unraveled specific biomarkers for several of these entities (PNPO deficiency, ALDH7A1 deficiency, ALDH4A1 deficiency, ALPL deficiency causing congenital hypophosphatasia and glycosylphosphatidylinositol anchoring defects with hyperphosphatasia), that can be detected in plasma or urine, while there is no biomarker to test for PLPHP deficiency. Secondary elevation of glycine or lactate was recognized as diagnostic pitfall. An algorithm for a standardized trial with vitamin B should be in place in every newborn unit in order not to miss these well-treatable inborn errors of metabolism. The Komrower lecture of 2022 provided me with the opportunity to tell the story about the conundrums of research into vitamin B -dependent epilepsies that kept some surprises and many novel insights into pathomechanisms of vitamin metabolism. Every single step had benefits for the patients and families that we care for and advocates for a close collaboration of clinician scientists with basic research.
Topics: Infant, Newborn; Humans; Vitamin B 6; Pyridoxine; Pyridoxal Phosphate; Epilepsy; Biomarkers; Vitamins
PubMed: 37428623
DOI: 10.1002/jimd.12655 -
Journal of the American Chemical Society Jun 2023Coenzymes are involved in ≥30% of enzymatic reactions and likely predate enzymes, going back to prebiotic chemistry. However, they are considered poor organocatalysts,...
Coenzymes are involved in ≥30% of enzymatic reactions and likely predate enzymes, going back to prebiotic chemistry. However, they are considered poor organocatalysts, and thus their pre-enzymatic function remains unclear. Since metal ions are known to catalyze metabolic reactions in the absence of enzymes, here we explore the influence of metal ions on coenzyme catalysis under conditions relevant to the origin of life (20-75 °C, pH 5-7.5). Specifically, Fe or Al, the two most abundant metals in the Earth's crust, were found to exhibit substantial cooperative effects in transamination reactions catalyzed by pyridoxal (PL), a coenzyme scaffold used by roughly 4% of all enzymes. At 75 °C and 7.5 mol % loading of PL/metal ion, Fe-PL was found to be 90-fold faster at catalyzing transamination than PL alone and 174-fold faster than Fe alone, whereas Al-PL was 85-fold faster than PL alone and 38-fold faster than Al alone. Under milder conditions, reactions catalyzed by Al-PL were >1000 times faster than those catalyzed by PL alone. Pyridoxal phosphate (PLP) exhibited similar behavior to PL. Experimental and theoretical mechanistic studies indicate that the rate-determining step in the PL-metal-catalyzed transamination is different from metal-free and biological PL-based catalysis. Metal coordination to PL lowers the p of the PL-metal complex by several units and slows the hydrolysis of imine intermediates by up to 259-fold. Coenzymes, specifically pyridoxal derivatives, could have exhibited useful catalytic function even before enzymes.
Topics: Pyridoxal; Pyridoxal Phosphate; Metals; Coenzymes; Amination; Catalysis
PubMed: 37278531
DOI: 10.1021/jacs.3c03542 -
Calcified Tissue International Jun 2020Low serum alkaline phosphatase (sALP)-hypophosphatasemia-is a characteristic of hypophosphatasia (HPP), but related to several clinical conditions. Here, we evaluated...
Low serum alkaline phosphatase (sALP)-hypophosphatasemia-is a characteristic of hypophosphatasia (HPP), but related to several clinical conditions. Here, we evaluated the frequency, persistency and the etiology of hypophosphatasemia in children. In retrospective analyses of sALP measurements from children, evaluated according to in-house constructed age- and sex-specific reference ranges, patients with no normal sALP measurement (Unresolved hypophosphatasemia) were invited for reanalysis. Prospectively, ALP substrates, pyridoxal-5-phosphate (PLP), and phosphoethanolamine (PEA) were measured in patients with persistent hypophosphatasemia. Radiographs and ALPL gene sequencing for HPP were performed to the cases with elevated PEA and/or PLP. From 130,340 sALP measurements of 93,162 patients, hypophosphatasemia was detected in 1404 samples from 867 patients (0.9%). Among them, 745 had at least one normal sALP values in laboratory records, grouped as transient hypophosphatasemia. 75 out of 122 patients with unresolved hypophosphatasemia could be reanalyzed for sALP, of whom PLP and PEA measurements were required in 37 due to persistent hypophosphatasemia. Both PEA and PLP were elevated in 4 patients, and ALPL gene analysis showed heterozygous mutations in 3 patients and homozygous in 1 patient. Elevated PEA with normal PLP were detected in 3 patients, and one had a heterozygous ALPL mutation. Anemia was the most common diagnosis, and upper respiratory tract infections and chronic diseases were more common in transient and unresolved hypophosphatasemia, respectively. In conclusion, reflected persistent hypophosphatasemia frequency was 1/1552 (0.06%) in this large pediatric cohort and, ALPL gene mutations were detected in 13.5% (5/37) of the studied cases. Although biochemical hypophosphatasemia is not uncommon, clinically significant HPP is rare.
Topics: Alkaline Phosphatase; Child; Ethanolamines; Female; Heterozygote; Humans; Hypophosphatasia; Male; Pyridoxal Phosphate; Retrospective Studies
PubMed: 32088736
DOI: 10.1007/s00223-020-00677-4 -
The Journal of Biological Chemistry 2021Pyridoxal 5'-phosphate (PLP), the catalytically active form of vitamin B, plays a pivotal role in metabolism as an enzyme cofactor. PLP is a very reactive molecule and...
Pyridoxal 5'-phosphate (PLP), the catalytically active form of vitamin B, 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).
Topics: Allosteric Site; Crystallography, X-Ray; Escherichia coli; Escherichia coli Infections; Escherichia coli Proteins; Humans; Models, Molecular; Protein Conformation; Pyridoxal Phosphate; Pyridoxaminephosphate Oxidase
PubMed: 34019876
DOI: 10.1016/j.jbc.2021.100795