Molecular mechanisms of the non-coenzyme action of thiamin in brain. Biochemical, structural and pathway analysis

Thiamin (vitamin B1) is a pharmacological agent boosting central metabolism through the action of the coenzyme thiamin diphosphate (ThDP). However, positive effects, including improved cognition, of high thiamin doses in neurodegeneration may be observed without increased ThDP or ThDPdependent enzymes in brain. Here, we determine protein partners and metabolic pathways where thiamin acts beyond its coenzyme role. Malate dehydrogenase, glutamate dehydrogenase and pyridoxal kinase were identified as abundant proteins binding to thiamin- or thiazolium-modified sorbents. Kinetic studies, supported by structural analysis, revealed allosteric regulation of these proteins by thiamin and/or its derivatives. Thiamin triphosphate and adenylated thiamin triphosphate activate glutamate dehydrogenase. Thiamin and ThDP regulate malate dehydrogenase isoforms and pyridoxal kinase. Thiamin regulation of enzymes related to malate-aspartate shuttle may impact on malate/citrate exchange, responsible for exporting acetyl residues from mitochondria. Indeed, bioinformatic analyses found an association between thiamin- and thiazolium-binding proteins and the term acetylation. Our interdisciplinary study shows that thiamin is not only a coenzyme for acetyl-CoA production, but also an allosteric regulator of acetyl-CoA metabolism including regulatory acetylation of proteins and acetylcholine biosynthesis. Moreover, thiamin action in neurodegeneration may also involve neurodegeneration-related 14-3-3, DJ-1 and β-amyloid precursor proteins identified among the thiamin- and/or thiazolium-binding proteins

A gene-nutrient interaction between vitamin B6 and serine hydroxymethyltransferase (SHMT) affects genome integrity in Drosophila

Pyridoxal 5'-phosphate (PLP), the catalytically active form of vitamin B6, participates as a cofactor to one carbon (1C) pathway that produces precursors for DNA metabolism. The concerted action of PLP-dependent serine hydroxymethyltransferase (SHMT) and thymidylate synthase (TS) leads to the biosynthesis of thymidylate (dTMP), which plays an essential function in DNA synthesis and repair. PLP deficiency causes chromosome aberrations (CABs) in Drosophila and human cells, rising the hypothesis that an altered 1C metabolism may be involved. To test this hypothesis, we used Drosophila as a model system and found, firstly, that in PLP deficient larvae SHMT activity is reduced by 40%. Second, we found that RNAi-induced SHMT depletion causes chromosome damage rescued by PLP supplementation and strongly exacerbated by PLP depletion. RNAi-induced TS depletion causes severe chromosome damage, but this is only slightly enhanced by PLP depletion. dTMP supplementation rescues CABs in both PLP-deficient and PLP-proficient SHMTRNAi . Altogether these data suggest that a reduction of SHMT activity caused by PLP deficiency contributes to chromosome damage by reducing dTMP biosynthesis. In addition, our work brings to light a gene-nutrient interaction between SHMT decreased activity and PLP deficiency impacting on genome stability that may be translated to human

Functional and structural properties of pyridoxal reductase (PdxI) from Escherichia coli. A pivotal enzyme in the vitamin B6 salvage pathway

Pyridoxine 4-dehydrogenase (PdxI), a NADPH-dependent pyridoxal reductase, is one of the key players in the Escherichia coli pyridoxal 5'-phosphate (PLP) salvage pathway. This enzyme, which catalyses the reduction of pyridoxal into pyridoxine, causes pyridoxal to be converted into PLP via the formation of pyridoxine and pyridoxine phosphate. The structural and functional properties of PdxI were hitherto unknown, preventing a rational explanation of how and why this longer, detoured pathway occurs, given that, in E. coli, two pyridoxal kinases (PdxK and PdxY) exist that could convert pyridoxal directly into PLP. Here, we report a detailed characterisation of E. coli PdxI that explains this behaviour. The enzyme efficiently catalyses the reversible transformation of pyridoxal into pyridoxine, although the reduction direction is thermodynamically strongly favoured, following a compulsory-order ternary-complex mechanism. In vitro, the enzyme is also able to catalyse PLP reduction and use NADH as an electron donor, although with lower efficiency. As with all members of the aldo-keto reductase (AKR) superfamily, the enzyme has a TIM barrel fold; however, it shows some specific features, the most important of which is the presence of an Arg residue that replaces the catalytic tetrad His residue that is present in all AKRs and appears to be involved in substrate specificity. The above results, in conjunction with kinetic and static measurements of vitamins B6 in cell extracts of E. coli wild-type and knockout strains, shed light on the role of PdxI and both kinases in determining the pathway followed by pyridoxal in its conversion to PLP, which has a precise regulatory function

Use of the Secreted Proteome of Trametes versicolor for Controlling the Cereal Pathogen Fusarium langsethiae

Fusarium langsethiae is amongst the most recently discovered pathogens of small grains cereals. F. langsethiae is the main producer, in Europe, of T2 and HT-toxins in small grain cereals, albeit often asymptomatic; this makes its control challenging. The European Union (EU) is pushing hard on the use of biocontrol agents to minimize the use of fungicides and pesticides, which are detrimental to the environment and responsible for serious pollution of the soil and superficial water. In line with EU directives (e.g., 128/2009), here we report the use of protein fractions, purified from the culture filtrate of the basidiomycete Trametes versicolor, for controlling F. langsethiae. T. versicolor, a so-called medicinal mushroom which is applied as a co-adjuvant in oncology and other pathologies as a producer of biological response modifiers. In this study, the exo-proteome of T. versicolor proved highly efficient in inhibiting the growth of F. langsethiae and the biosynthesis of the T2 toxin. Results are promising for its future use as a sustainable product to control F. langsethiae infection in cereals under field conditions

Inhibition of human pyridoxal kinase by 2-acetyl-4-((1R,2S,3R)-1,2,3,4-1 tetrahydroxybutyl)-imidazole (THI)

2-Acetyl-4-((1R,2S,3R)-1,2,3,4-tetrahydroxybutyl)imidazole (THI) is observed as a minor contaminant in caramel food colourings (E 150c). Feeding experiments with rodents have revealed a significant lymphopenic effect that has been linked to the presence of THI in these food colourings. Pyridoxal kinase inhibition by THI has been suggested, but not demonstrated, as a mode of action as it leads to lowered levels of pyridoxal-5’-phosphate, which are known to cause lymphopenia. Recently, THI was also shown to inhibit sphingosine-1-phosphate lyase causing comparable immunosuppressive effects and derivatives of THI are being developed for the treatment of rheumatoid arthritis in humans. Interestingly, sphingosine-1-phosphate lyase activity depends on pyridoxal-5’-phosphate, which in turn is provided by pyridoxal kinase. This report shows that THI does inhibit pyridoxal kinase with competitive and mixed-type non-competitive behaviour towards its two substrates, pyridoxal and ATP, respectively. The corresponding inhibition constants are in the low millimolar range

The potential of plant-based bioactive compounds on inhibition of aflatoxin B1 biosynthesis and down-regulation of aflR, aflM and aflP genes

The use of plant extracts in pre- and post-harvest disease management of agricultural crops to cope with aflatoxin B1 contamination has shown great promise due to their capability in managing toxins and safe-keeping the quality. We investigated the anti-aflatoxigenic effect of multiple doses of eight plant extracts (Heracleum persicum, Peganum harmala, Crocus sativus, Trachyspermum ammi, Rosmarinus officinalis, Anethum graveolens, Berberis vulgaris, Berberis thunbergii) on Aspergillus flavus via LC-MS and the down-regulatory effect of them on aflR, aflM and aflP genes involved in the aflatoxin B1 biosynthesis pathway using RT-qPCR analyses. Our results showed that H. persicum (4 mg/mL), P. harmala (6 mg/mL) and T. ammi (2 mg/mL) completely stopped the production of aflatoxin B1, without inducing significant changes in A. flavus growth. Furthermore, our findings showed a highly significant correlation between the gene expression and the aflatoxin B1 biosynthesis, such that certain doses of the extracts reduced or blocked the expression of the aflR, aflM and aflP and consequently reduced the synthesis of aflatoxin B1. Interestingly, compared to the regulatory gene (aflR), the down-regulation of expression in the structural genes (aflM and aflP) was more consistent and correlated with the inhibition of aflatoxin B1 production. Overall, this study reveals the anti-aflatoxigenic mechanisms of the selected plant extracts at the gene expression level and provides evidence for their use in plant and crop protection

Phytochemical analysis of Linaria purpurea (L.) Mill. and inhibitory activity on the production of aflatoxin B1 (AFB1) in Aspergillus flavus Link. of one of its metabolites, antirrhinoside

In this work, the first phytochemical study on the total polar fraction of Linaria purpurea (L.) Mill. was performed by means of column chromatography and NMR and MS analysis. Seven compounds were identified i.e. pheophytin a (1), methyl-pheophorbide a (2), linaride (3), antirrhide (4), antirrhinoside (5), linarioside (6) and shikimic acid (7), belonging to three different classes of natural compounds. Compound (2) represents a new compound for the family as well as compound (1) is for the genus and compounds (3, 6, 7) are for the species. The chemosystematic and ethnopharmacological relevance of these results were widely discussed. In addition, compound (5) was tested, for the first time, for its potential antifungal activity, showing to drastically reduce the production of aflatoxin B1 in Aspergillus flavus Link. This further property of compound (5) makes it a potential natural and green anti-aflatoxin B1 agent to be used in the food industry

Molecular mechanisms of the non-coenzyme action of thiamin in brain: biochemical, structural and pathway analysis.

Thiamin (vitamin B1) is a pharmacological agent boosting central metabolism through the action of the coenzyme thiamin diphosphate (ThDP). However, positive effects, including improved cognition, of high thiamin doses in neurodegeneration may be observed without increased ThDP or ThDP-dependent enzymes in brain. Here, we determine protein partners and metabolic pathways where thiamin acts beyond its coenzyme role. Malate dehydrogenase, glutamate dehydrogenase and pyridoxal kinase were identified as abundant proteins binding to thiamin- or thiazolium-modified sorbents. Kinetic studies, supported by structural analysis, revealed allosteric regulation of these proteins by thiamin and/or its derivatives. Thiamin triphosphate and adenylated thiamin triphosphate activate glutamate dehydrogenase. Thiamin and ThDP regulate malate dehydrogenase isoforms and pyridoxal kinase. Thiamin regulation of enzymes related to malate-aspartate shuttle may impact on malate/citrate exchange, responsible for exporting acetyl residues from mitochondria. Indeed, bioinformatic analyses found an association between thiamin- and thiazolium-binding proteins and the term acetylation. Our interdisciplinary study shows that thiamin is not only a coenzyme for acetyl-CoA production, but also an allosteric regulator of acetyl-CoA metabolism including regulatory acetylation of proteins and acetylcholine biosynthesis. Moreover, thiamin action in neurodegeneration may also involve neurodegeneration-related 14-3-3, DJ-1 and β-amyloid precursor proteins identified among the thiamin- and/or thiazolium-binding proteins

Structural stability of cold-adapted serine hydroxymethyltransferase, a tool for ß-hydroxy-α-amino acid biosynthesis

Serine hydroxymethyltransferase (SHMT) is a pyridoxal-5'-phosphate-dependent enzyme that catalyses the reversible conversion of l-serine and tetrahydropteroylglutamate to glycine and 5,10-methylenetetrahydropteroylglutamate. This enzyme represents a good model for analysing the intricate relationship between activity and stability, since it is ubiquitous in nature and well characterized from different organisms. Besides its physiological role, SHMT catalyses the reversible cleavage of several beta-hydroxy amino acids varying in substituent and stereochemistry at C-beta and, for this reason, it represents a good tool for biotechnological applications. SHMT from the psychrophilic bacterium Psychromonas ingrahamii (piSHMT) displays the interesting feature of having high specific activity in the cleavage of beta-hydroxy amino acids at all temperatures. In the present study, we compare the temperature dependence of psychrophilic piSHMT and mesophilic Escherichia coli SHMT (ecSHMT) in catalysing the physiological hydroxymethyltransferase reaction. We also investigate the structural stability of both enzymes by performing equilibrium unfolding experiments. Unexpectedly, our results show that piSHMT is a less efficient catalyst than ecSHMT in the hydroxymethyltransferase activity at all temperatures. Moreover, the two enzymes have comparable structural stability, with piSHMT showing even higher resistance to chemical denaturation by urea and to inactivation by formaldehyde. This unusual structural stability of piSHMT and its high efficiency at low temperatures as catalyst of beta-hydroxy amino acids cleavage make this enzyme an attractive tool for industrial applications

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