PFOA accumulation in the leaves of basil (Ocimum basilicum L.) and its effects on plant growth, oxidative status, and photosynthetic performance.

BACKGROUND: Perfluoroalkyl substances (PFASs) are emerging contaminants of increasing concern due to their presence in the environment, with potential impacts on ecosystems and human health. These substances are considered "forever chemicals" due to their recalcitrance to degradation, and their accumulation in living organisms can lead to varying levels of toxicity based on the compound and species analysed. Furthermore, concerns have been raised about the possible transfer of PFASs to humans through the consumption of edible parts of food plants. In this regard, to evaluate the potential toxic effects and the accumulation of perfluorooctanoic acid (PFOA) in edible plants, a pot experiment in greenhouse using three-week-old basil (Ocimum basilicum L.) plants was performed adding PFOA to growth substrate to reach 0.1, 1, and 10 mg Kg- 1 dw. RESULTS: After three weeks of cultivation, plants grown in PFOA-added substrate accumulated PFOA at different levels, but did not display significant differences from the control group in terms of biomass production, lipid peroxidation levels (TBARS), content of α-tocopherol and activity of ascorbate peroxidase (APX), catalase (CAT) and guaiacol peroxidase (POX) in the leaves. A reduction of total phenolic content (TPC) was instead observed in relation to the increase of PFOA content in the substrate. Furthermore, chlorophyll content and photochemical reflectance index (PRI) did not change in plants exposed to PFAS in comparison to control ones. Chlorophyll fluorescence analysis revealed an initial, rapid photoprotective mechanism triggered by PFOA exposure, with no impact on other parameters (Fv/Fm, ΦPSII and qP). Higher activity of glutathione S-transferase (GST) in plants treated with 1 and 10 mg Kg- 1 PFOA dw (30 and 50% to control, respectively) paralleled the accumulation of PFOA in the leaves of plants exposed to different PFOA concentration in the substrate (51.8 and 413.9 ng g- 1 dw, respectively). CONCLUSION: Despite of the absorption and accumulation of discrete amount of PFOA in the basil plants, the analysed parameters at biometric, physiological and biochemical level in the leaves did not reveal any damage effect, possibly due to the activation of a detoxification pathway likely involving GST.

Thiamine-dependent regulation of mammalian brain pyridoxal kinase in vitro and in vivo.

Vitamins B1 (thiamine) and B6 (pyridox (al/ine/amine)) are crucial for central nervous system (CNS) function and neurogenesis due to the coenzyme action of their phosphorylated derivatives in the brain metabolism of glucose and neurotransmitters. Here, the non-coenzyme action of thiamine on the major mammalian producers of pyridoxal-5'-phosphate (PLP), such as pyridoxal kinase (PdxK) and pyridoxine 5'-phosphate oxidase (PNPO), is characterized. Among the natural thiamine compounds, thiamine triphosphate (ThTP) is the best effector of recombinant human PdxK (hPdxK) in vitro, inhibiting hPdxK in the presence of Mg2+ but activating the Zn2+ -dependent reaction. Inhibition of hPdxK by thiamine antagonists decreases from amprolium to pyrithiamine to oxythiamine, highlighting possible dysregulation of both the B1 - and B6 -dependent metabolism in the chemical models of thiamine deficiency. Compared with the canonical hPdxK, the D87H and V128I variants show a twofold increase in Kapp of thiamine inhibition, and the V128I and H246Q variants show a fourfold and a twofold decreased Kapp of thiamine diphosphate (ThDP), respectively. Thiamine administration changes diurnal regulation of PdxK activity and phosphorylation at Ser213 and Ser285, expression of the PdxK-related circadian kinases/phosphatases in the rat brain, and electrocardiography (ECG). In contrast to PdxK, PNPO is not affected by thiamine or its derivatives, either in vitro or in vivo. Dephosphorylation of the PdxK Ser285, potentially affecting mobility of the ATP-binding loop, inversely correlates with the enzyme activity. Dephosphorylation of the PdxK Ser213, which is far away from the active site, does not correlate with the activity. The correlations analysis suggests the PdxK Ser213 to be a target of kinase MAP2K1 and phosphatase Ppp1ca. Diurnal effects of thiamine administration on the metabolically linked ThDP- and PLP-dependent enzymes may support the brain homeostatic mechanisms and physiological fitness.

Allosteric feedback inhibition of pyridoxine 5'-phosphate oxidase from Escherichia coli.

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.

Pyridoxine-5'-phosphate oxidase (Pnpo) deficiency: Clinical and biochemical alterations associated with the C.347g>A (P.·Arg116gln) mutation.

BACKGROUND: Pyridoxal-5'-phosphate oxidase (PNPO) deficiency presents as a severe neonatal encephalopathy responsive to pyridoxal-5'-phosphate (PLP) or pyridoxine. Recent studies widened the phenotype of this condition and detected genetic variants on PNPO gene whose pathogenic role and clinical expression remain to be established. OBJECTIVE: This paper aims to characterize the functional effects of the c.347G>A (p.Arg116Gln) mutation in the PNPO gene in order to define its pathogenicity and describe the clinical features of new patients with epilepsy carrying this mutation. METHODS: Arg116Gln protein variant was expressed as recombinant protein. The mutant protein was characterized with respect to structural and kinetic properties, thermal stability, binding constants of cofactor (FMN) and product (PLP). We also reviewed clinical data of 3 new patients carrying the mutation. RESULTS: The Arg116Gln mutation does not alter the overall enzyme structure and only slightly affects its catalytic efficiency; nevertheless, this mutation affects thermal stability of PNPO, reduces its affinity for FMN and impairs transfer of PLP to PLP-dependent enzymes. Three boys with seizure onset between 8months and 3years of age, carrying the Arg116Gln mutation, are described. These three patients exhibited different seizure types associated with interictal EEG abnormalities and slow background activity. Mild/moderate intellectual disability was observed in 2/3 patients. A dramatic therapeutic response to pyridoxine was observed in the only patient who still had active seizures when starting treatment, while in all three patients interictal EEG discharges and background activity improved after pyridoxine treatment was initiated. CONCLUSIONS: The reported data support a pathogenic role of the c.347G>A (p.Arg116Gln) mutation in PNPO deficiency. The later onset of symptoms and the milder epilepsy phenotype of these expand the disease phenotype.

On the mechanism of Escherichia coli pyridoxal kinase inhibition by pyridoxal and pyridoxal 5'-phosphate.

Pyridoxal 5'-phosphate (PLP), the catalytically active form of vitamin B6, plays a crucial role in several cellular processes. In most organisms, PLP is recycled from nutrients and degraded B6-enzymes in a salvage pathway that involves pyridoxal kinase (PLK), pyridoxine phosphate oxidase and phosphatase activities. Regulation of the salvage pathway is poorly understood. Escherichia coli possesses two distinct pyridoxal kinases, PLK1, which is the focus of the present work, and PLK2. From previous studies dating back to thirty years ago, pyridoxal (PL) was shown to inhibit E. coli PLK1 forming a covalent link with the enzyme. This inhibition was proposed to play a regulative role in vitamin B6 metabolism, although its details had never been clarified. Recently, we have shown that also PLP produced during PLK1 catalytic cycle acts as an inhibitor, forming a Schiff base with Lys229, without being released in the solvent. The question arises as to which is the actual inhibition mechanism by PL and PLP. In the present work, we demonstrated that also PL binds to Lys229 as a Schiff base. However, the isolated covalent PLK1-PL complex is not inactive but, in the presence of ATP, is able to catalyse the single turnover production of PLP, which binds tightly to the enzyme and is ultimately responsible for its inactivation. The inactivation mechanism mediated by Lys229 may play a physiological role in controlling cellular levels of PLP. This article is part of a Special Issue entitled: Cofactor-dependent proteins: evolution, chemical diversity and bio-applications.

Insights into the substrate specificity, structure, and dynamics of plant histidinol-phosphate aminotransferase (HISN6).

Histidinol-phosphate aminotransferase is the sixth protein (hence HISN6) in the histidine biosynthetic pathway in plants. HISN6 is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes the reversible conversion of imidazole acetol phosphate into L-histidinol phosphate (HOLP). Here, we show that plant HISN6 enzymes are closely related to the orthologs from Chloroflexota. The studied example, HISN6 from Medicago truncatula (MtHISN6), exhibits a surprisingly high affinity for HOLP, which is much higher than reported for bacterial homologs. Moreover, unlike the latter, MtHISN6 does not transaminate phenylalanine. High-resolution crystal structures of MtHISN6 in the open and closed states, as well as the complex with HOLP and the apo structure without PLP, bring new insights into the enzyme dynamics, pointing at a particular role of a string-like fragment that oscillates near the active site and participates in the HOLP binding. When MtHISN6 is compared to bacterial orthologs with known structures, significant differences arise in or near the string region. The high affinity of MtHISN6 appears linked to the particularly tight active site cavity. Finally, a virtual screening against a library of over 1.3 mln compounds revealed three sites in the MtHISN6 structure with the potential to bind small molecules. Such compounds could be developed into herbicides inhibiting plant HISN6 enzymes absent in animals, which makes them a potential target for weed control agents.

Cultivation of Melilotus officinalis as a source of bioactive compounds in association with soil recovery practices.

Introduction: Melilotus officinalis is a Leguminosae with relevant applications in medicine and soil recovery. This study reports the application of Melilotus officinalis plants in soil recovery and as a source of bioactive compounds. Methods: Plants were cultivated in semiarid soil under four different fertilizer treatments, urban waste compost at 10 t/ha and 20 t/ha, inorganic fertilizer and a control (no fertilizer). Agronomic properties of soil (pH, EC, soil respiration, C content, macro- and microelements) were analyzed before and after treatment. Also, germination, biomass, element contents, and physiological response were evaluated. Metabolite composition of plants was analyzed through Fourier-transform ion cyclotron resonance mass spectrometry (FT-ICR MS). Results and discussion: Results showed a significant enhancement of the soil microbial activity in planted soils amended with compost, though there were no other clear effects on the soil physicochemical and chemical characteristics during the short experimental period. An improvement in M. officinalis germination and growth was observed in soils with compost amendment. Metabolite composition of plants was analyzed through Fourier-transform ion cyclotron resonance mass spectrometry (FT-ICR MS). Principal Component and Agglomerative Hierarchical Clustering models suggest that there is a clear separation of the metabolome of four groups of plants grown under different soil treatments. The five most important discriminative metabolites (annotated) were oleamide, palmitic acid, stearic acid, 3-hydroxy-cis-5-octenoylcarnitine, and 6-hydroxynon-7- enoylcarnitine. This study provides information on how the metabolome of Melilotus might be altered by fertilizer application in poor soil regions. These metabolome changes might have repercussions for the application of this plant in medicine and pharmacology. The results support the profitability of Melilotus officinalis cultivation for bioactive compounds production in association with soil recovery practices.

Increased thermostability of thylakoid membranes in isoprene-emitting leaves probed with three biophysical techniques.

Three biophysical approaches were used to get insight into increased thermostability of thylakoid membranes in isoprene-emittingplants.Arabidopsis (Arabidopsis thaliana) plants genetically modified to make isoprene and Platanus orientalis leaves, in which isoprene emission was chemically inhibited, were used. First, in the circular dichroism spectrum the transition temperature of the main band at 694 nm was higher in the presence of isoprene, indicating that the heat stability of chiral macrodomains of chloroplast membranes, and specifically the stability of ordered arrays of light-harvesting complex II-photosystem II in the stacked region of the thylakoid grana, was improved in the presence of isoprene. Second, the decay of electrochromic absorbance changes resulting from the electric field component of the proton motive force (ΔA515) was evaluated following single-turnover saturating flashes. The decay of ΔA515 was faster in the absence of isoprene when leaves of Arabidopsis and Platanus were exposed to high temperature, indicating that isoprene protects the thylakoid membranes against leakiness at elevated temperature. Finally, thermoluminescence measurements revealed that S2Q(B)⁻ charge recombination was shifted to higher temperature in Arabidopsis and Platanus plants in the presence of isoprene, indicating higher activation energy for S2Q(B)⁻ redox pair, which enables isoprene-emitting plants to perform efficient primary photochemistry of photosystem II even at higher temperatures. The data provide biophysical evidence that isoprene improves the integrity and functionality of the thylakoid membranes at high temperature. These results contribute to our understanding of isoprene mechanism of action in plant protection against environmental stresses.

The impact of root temperature on photosynthesis and isoprene emission in three different plant species.

Most of the perennial plant species, particularly trees, emit volatile organic compounds (BVOCs) such as isoprene and monoterpenes, which in several cases have been demonstrated to protect against thermal shock and more generally against oxidative stress. In this paper, we show the response of three strong isoprene emitter species, namely, Phragmites australis, Populus x euramericana, and Salix phylicifolia exposed to artificial or natural warming of the root system in different conditions. This aspect has not been investigated so far while it is well known that warming the air around a plant stimulates considerably isoprene emission, as also shown in this paper. In the green house experiments where the warming corresponded with high stress conditions, as confirmed by higher activities of the main antioxidant enzymes, we found that isoprene uncoupled from photosynthesis at a certain stage of the warming treatment and that even when photosynthesis approached to zero isoprene emission was still ongoing. In the field experiment, in a typical cold-limited environment, warming did not affect isoprene emission whereas it increased significantly CO2 assimilation. Our findings suggest that the increase of isoprene could be a good marker of heat stress, whereas the decrease of isoprene a good marker of accelerated foliar senescence, two hypotheses that should be better investigated in the future.

Arabidopsis thaliana serine hydroxymethyltransferases: functions, structures, and perspectives.

Serine hydroxymethyltransferase (SHM) is one of the hallmarks of one-carbon metabolism. In plants, isoforms of SHM participate in photorespiration and/or transfer the one-carbon unit from L-serine to tetrahydrofolate (THF), hence producing 5,10-CH2-THF that is needed, e.g., for biosynthesis of methionine, thymidylate, and purines. These links highlight the importance of SHM activity in DNA biogenesis, its epigenetic methylations, and in stress responses. Plant genomes encode several SHM isoforms that localize to cytosol, mitochondria, plastids, and nucleus. In this work, we present a thorough functional and structural characterization of all seven SHM isoforms from Arabidopsis thaliana (AtSHM1-7). In particular, we analyzed tissue-specific expression profiles of the AtSHM genes. We also compared catalytic properties of the active AtSHM1-4 in terms of catalytic efficiency in both directions and inhibition by the THF substrate. Despite numerous attempts to rescue the SHM activity of AtSHM5-7, we failed, which points towards different physiological functions of these isoforms. Comparative analysis of experimental and predicted three-dimensional structures of AtSHM1-7 proteins indicated differences in regions that surround the entrance to the active site cavity.

Physiological and Biochemical Markers of the Sex-Specific Sensitivity to Epileptogenic Factors, Delayed Consequences of Seizures and Their Response to Vitamins B1 and B6 in a Rat Model.

The disturbed metabolism of vitamins B1 or B6, which are essential for neurotransmitters homeostasis, may cause seizures. Our study aims at revealing therapeutic potential of vitamins B1 and B6 by estimating the short- and long-term effects of their combined administration with the seizure inductor pentylenetetrazole (PTZ). The PTZ dose dependence of a seizure and its parameters according to modified Racine's scale, along with delayed physiological and biochemical consequences the next day after the seizure are assessed regarding sexual dimorphism in epilepsy. PTZ sensitivity is stronger in the female than the male rats. The next day after a seizure, sex differences in behavior and brain biochemistry arise. The induced sex differences in anxiety and locomotor activity correspond to the disappearance of sex differences in the brain aspartate and alanine, with appearance of those in glutamate and glutamine. PTZ decreases the brain malate dehydrogenase activity and urea in the males and the phenylalanine in the females. The administration of vitamins B1 and B6 24 h before PTZ delays a seizure in female rats only. This desensitization is not observed at short intervals (0.5-2 h) between the administration of the vitamins and PTZ. With the increasing interval, the pyridoxal kinase (PLK) activity in the female brain decreases, suggesting that the PLK downregulation by vitamins contributes to the desensitization. The delayed effects of vitamins and/or PTZ are mostly sex-specific and interacting. Our findings on the sex differences in sensitivity to epileptogenic factors, action of vitamins B1/B6 and associated biochemical events have medical implications.

Molecular characterization of pyridoxine 5'-phosphate oxidase and its pathogenic forms associated with neonatal epileptic encephalopathy.

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.

Structural and kinetic properties of serine hydroxymethyltransferase from the halophytic cyanobacterium Aphanothece halophytica provide a rationale for salt tolerance.

Serine hydroxymethyltransferase (SHMT) is a pyridoxal 5'-phosphate-dependent enzyme that plays a pivotal role in cellular one­carbon metabolism. In plants and cyanobacteria, this enzyme is also involved in photorespiration and confers salt tolerance, as in the case of SHMT from the halophilic cyanobacterium Aphanothece halophytica (AhSHMT). We have characterized the catalytic properties of AhSHMT in different salt and pH conditions. Although the kinetic properties of AhSHMT correlate with those of the mesophilic orthologue from Escherichia coli, AhSHMT appears more catalytically efficient, especially in presence of salt. Our studies also reveal substrate inhibition, previously unobserved in AhSHMT. Furthermore, addition of the osmoprotectant glycine betaine under salt conditions has a distinct positive effect on AhSHMT activity. The crystal structures of AhSHMT in three forms, as internal aldimine, as external aldimine with the l-serine substrate, and as a covalent complex with malonate, give structural insights on the possible role of specific amino acid residues implicated in the halophilic features of AhSHMT. Importantly, we observed that overexpression of the gene encoding SHMT, independently from its origin, increases the capability of E. coli to grow in high salt conditions, suggesting that the catalytic activity of this enzyme in itself plays a fundamental role in salt tolerance.

Effects of Soil Fertilization on Terpenoids and Other Carbon-Based Secondary Metabolites in Rosmarinus officinalis Plants: A Comparative Study.

Rosmarinus officinalis is an evergreen aromatic plant with important commercial interest as it contains numerous essential oils (composed of terpenoid compounds) and phenolic constituents (natural antioxidant compounds). This work aims at evaluating the concomitant effects of different inorganic and organic fertilization treatments and the subsequent increases in soil nutrient availability on terpenoids and other carbon-based secondary metabolites, e.g., flavonoids and phenolic compounds, in Rosmarinus officinalis leaves. The results showed that, as expected, the structural carbohydrate content (lignocellulosic compounds) in stems was higher in fertilized plants than in controls. Additionally, positive correlations were observed of the absolute amounts of total terpenoids and some single terpenoid compounds with N or P contents in leaves. On the contrary, the phenolic and flavonoid concentrations in all the rosemary plant parts were lower with the fertilization treatments. Indeed, negative correlations between the phenolic compounds (and flavonoids) and N in rosemary leaves were also found. Overall, the results suggest that the terpenoid production's response to fertilization was due to N, which is essential for protein synthesis and terpene synthase activity, and to P, which is necessary for the synthesis of both terpenoid precursors and ATP and NADPH, also needed for terpenoid synthesis. On the other hand, the basis for the fertilization's effects on the production of phenolic compounds is the direct nitrogen trade-off between growth and the shikimic acid pathway by which phenolics compounds are synthesized.

Structural basis of methotrexate and pemetrexed action on serine hydroxymethyltransferases revealed using plant models.

Serine hydroxymethyltransferases (SHMTs) reversibly transform serine into glycine in a reaction accompanied with conversion of tetrahydrofolate (THF) into 5,10-methylene-THF (5,10-meTHF). In vivo, 5,10-meTHF is the main carrier of one-carbon (1C) units, which are utilized for nucleotide biosynthesis and other processes crucial for every living cell, but hyperactivated in overproliferating cells (e.g. cancer tissues). SHMTs are emerging as a promising target for development of new drugs because it appears possible to inhibit growth of cancer cells by cutting off the supply of 5,10-meTHF. Methotrexate (MTX) and pemetrexed (PTX) are two examples of antifolates that have cured many patients over the years but target different enzymes from the folate cycle (mainly dihydrofolate reductase and thymidylate synthase, respectively). Here we show crystal structures of MTX and PTX bound to plant SHMT isozymes from cytosol and mitochondria-human isozymes exist in the same subcellular compartments. We verify inhibition of the studied isozymes by a thorough kinetic analysis. We propose to further exploit antifolate scaffold in development of SHMT inhibitors because it seems likely that especially polyglutamylated PTX inhibits SHMTs in vivo. Structure-based optimization is expected to yield novel antifolates that could potentially be used as chemotherapeutics.

A Comprehensive Computational Analysis of Mycobacterium Genomes Pinpoints the Genes Co-occurring with YczE, a Membrane Protein Coding Gene Under the Putative Control of a MocR, and Predicts its Function.

Bacterial proteins belonging to the YczE family are predicted to be membrane proteins of yet unknown function. In many bacterial species, the yczE gene coding for the YczE protein is divergently transcribed with respect to an adjacent transcriptional regulator of the MocR family. According to in silico predictions, proteins named YczR are supposed to regulate the expression of yczE genes. These regulators linked to the yczE genes are predicted to constitute a subfamily within the MocR family. To put forward hypotheses amenable to experimental testing about the possible function of the YczE proteins, a phylogenetic profile strategy was applied. This strategy consists in searching for those genes that, within a set of genomes, co-occur exclusively with a certain gene of interest. Co-occurrence can be suggestive of a functional link. A set of 30 mycobacterial complete proteomes were collected. Of these, only 16 contained YczE proteins. Interestingly, in all cases each yczE gene was divergently transcribed with respect to a yczR gene. Two orthology clustering procedures were applied to find proteins co-occurring exclusively with the YczE proteins. The reported results suggest that YczE may be involved in the membrane translocation and metabolism of sulfur-containing compounds mostly in rapidly growing, low pathogenicity mycobacterial species. These observations may hint at potential targets for therapies to treat the emerging opportunistic infections provoked by the widespread environmental mycobacterial species and may contribute to the delineation of the genomic and physiological differences between the pathogenic and non-pathogenic mycobacterial species.

Biochemical data from the characterization of a new pathogenic mutation of human pyridoxine-5'-phosphate oxidase (PNPO).

PNPO deficiency is responsible of severe neonatal encephalopathy, responsive to pyridoxal-5'-phosphate (PLP) or pyridoxine. Recent studies widened the phenotype of this condition and detected new genetic variants on PNPO gene, whose pathogenetic role and clinical expression remain to be established. One of these mutations, Arg116Gln, is of particular interest because of its later onset of symptoms (beyond the first months of life) and its peculiar epileptic manifestations in patients. This protein variant was expressed as recombinant protein in E coli, purified to homogeneity, and characterized with respect to structural and kinetic properties, stability, binding constants of cofactor flavin mononucleotide (FMN) and product (PLP) in order to define the molecular and structural bases of its pathogenicity. For interpretation and discussion of reported data, together with the description of clinical studies, refer to the article [1] (doi: 10.1016/j.ymgme.2017.08.003).

Towards a new interaction enzyme:coenzyme.

Ferredoxin-NADP(+) reductase catalyses NADP(+) reduction, being specific for NADP(+)/H. To understand coenzyme specificity determinants and coenzyme specificity reversion, mutations at the NADP(+)/H pyrophosphate binding and of the C-terminal regions have been simultaneously introduced in Anabaena FNR. The T155G/A160T/L263P/Y303S mutant was produced. The mutated enzyme presents similar k(cat) values for NADPH and NADH, around 2.5 times slower than that reported for WT FNR with NADPH. Its K(m) value for NADH decreased 20-fold with regard to WT FNR, whereas the K(m) for NADPH remains similar. The combined effect is a much higher catalytic efficiency for NAD(+)/H, with a minor decrease of that for NADP(+)/H. In the mutated enzyme, the specificity for NADPH versus NADH has been decreased from 67,500 times to only 12 times, being unable to discriminate between both coenzymes. Additionally, giving the role stated for the C-terminal Tyr in FNR, its role in the energetics of the FAD binding has been analysed.

Molecular mechanism of PdxR ­ a transcriptional activator involved in the regulation of vitamin B6 biosynthesis in the probiotic bacterium Bacillus clausii.

Pyridoxal 5'-phosphate (PLP), the well-known active form of vitamin B6 , is an essential enzyme cofactor involved in a large number of metabolic processes. PLP levels need to be finely tuned in response to cell requirements; however, little is known about the regulation of PLP biosynthesis and recycling pathways. The transcriptional regulator PdxR activates transcription of the pdxST genes encoding PLP synthase. It is characterized by an N-terminal helix-turn-helix motif that binds DNA and an effector-binding C-terminal domain homologous to PLP-dependent enzymes. Although it is known that PLP acts as an anti-activator, the mechanism of action of PdxR is unknown. In the present study, we analyzed the biochemical and DNA-binding properties of PdxR from the probiotic Bacillus clausii. Spectroscopic measurements showed that PLP is the only B6 vitamer that acts as an effector molecule of PdxR. Binding of PLP to PdxR determines a protein conformational change, as detected by gel filtration chromatography and limited proteolysis experiments. We showed that two direct repeats and one inverted repeat are present in the DNA promoter region and PdxR is able to bind DNA fragments containing any combination of two of them. However, when PLP binds to PdxR, it modifies the DNA-binding properties of the protein, making it selective for inverted repeats. A molecular mechanism is proposed in which the two different DNA binding modalities of PdxR determined by the presence or absence of PLP are responsible for the control of pdxST transcription.