Deoxyxylulose Phosphate Research Articles

Enhanced Biochemical and Morphological Parameters in Eruca vesicaria by Applications of date-palm seed Extract

AbstractThe current global context stimulating agroecology and green agriculture need to explore for the novel sustainable and eco-conscious methods for safeguarding plants. The aim of this study is to exploit the rich contents of date palm seeds (DPS) as an appropriate organic elicitor to boost growth and plant secondary metabolism in Eruca vesicaria. The seeds of DPS were utilized in the extraction process using distilled water to create an aqueous extract, which underwent phytochemical characterization. In a pot experiment, Eruca vesicaria seeds were soaked in serial doses (0, 20, 40, 80, and 120 g l ⁻¹) of DPS aqueous extract, and the 21-days old seedlings were collected. DPS extract analysis indicated adequate P and N contents, antioxidant compounds, and exhibited antioxidant activity. The primary components identified in the analysis of DPS were 2-Dodecenal, fatty acids and cyclopentane butyl-acid. Priming with DPS extract significantly improved its bio-stimulating capacity by enhancing fresh and dry biomasses, photosynthetic pigments, and primary metabolites in response to the optimal DSP concentration of 80 g l⁻¹. Stress biomarkers (H2O2 and malondialdehyde (MDA)) were found in the ranges of water-primed control except for the highest dose of DPS extract (120 g l⁻¹). Further, priming with DPS extract increased the secondary metabolites (total phenolics and flavonoids) besides the activity of polyphenol oxidase (PPO), suggesting an enhanced cell redox system. The expression patterns of a series of specific key genes included in secondary metabolism modulation as, phenylalanine ammonia lyase (PAL), chalcone synthase (CHS), chalcone isomerase (CHI), flavonol synthase (FLS), and deoxyxylulose phosphate reductoisomerase (DXR), were activated following treatments with DPS extract. Overall, the present study underscores that the application of DPS extract can stimulate the growth and bioactive constituents of Eruca vesicaria, thus elevating its potential as a nutraceutical and medicinal value.

Enhancing bio-isoprene production in Escherichia coli through a combinatorial optimization approach

Enhancing bio-isoprene production in Escherichia coli through a combinatorial optimization approach

Deoxyxylulose 5-Phosphate Synthase Does Not Play a Major Role in Regulating the Methylerythritol 4-Phosphate Pathway in Poplar.

The plastidic 2-C-methylerythritol 4-phosphate (MEP) pathway supplies the precursors of a large variety of essential plant isoprenoids, but its regulation is still not well understood. Using metabolic control analysis (MCA), we examined the first enzyme of this pathway, 1-deoxyxylulose 5-phosphate synthase (DXS), in multiple grey poplar (Populus × canescens) lines modified in their DXS activity. Single leaves were dynamically labeled with 13CO2 in an illuminated, climate-controlled gas exchange cuvette coupled to a proton transfer reaction mass spectrometer, and the carbon flux through the MEP pathway was calculated. Carbon was rapidly assimilated into MEP pathway intermediates and labeled both the isoprene released and the IDP+DMADP pool by up to 90%. DXS activity was increased by 25% in lines overexpressing the DXS gene and reduced by 50% in RNA interference lines, while the carbon flux in the MEP pathway was 25-35% greater in overexpressing lines and unchanged in RNA interference lines. Isoprene emission was also not altered in these different genetic backgrounds. By correlating absolute flux to DXS activity under different conditions of light and temperature, the flux control coefficient was found to be low. Among isoprenoid end products, isoprene itself was unchanged in DXS transgenic lines, but the levels of the chlorophylls and most carotenoids measured were 20-30% less in RNA interference lines than in overexpression lines. Our data thus demonstrate that DXS in the isoprene-emitting grey poplar plays only a minor part in controlling flux through the MEP pathway.

Upregulated synthesis and production of bioactive compounds in Lotus arabicus L. by in vitro feeding with dried powder of date palm seeds

BackgroundPlants are considered the primary source of many principal bioactive compounds that have been utilized in a wide range of applications including the pharmaceutical and biotechnological industries. Therefore, there is an imperative need to modulate the production of natural bioactive components. The present study aimed to determine the importance of dried and pulverized date palm seeds (DPS) as a natural elicitor for the synthesis of secondary metabolites in Lotus arabicus L.ResultsThe presence of various antioxidant compounds, simple sugars, amino acids, fatty acids and reasonable mineral contents was distinct in the phytochemical characterization of DPS. The major components detected in DPS analysis were the 5-(hydroxymethyl) furfural and 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyranone. The induced callus of L. arabicus (seven weeks old) was supplemented with DPS at different concentrations (0, 2, 4, 8 and 10 g/l) in culture media. Treatment with 8 g/l DPS induced the highest antioxidant capacity, ascorbic acid content and secondary metabolites (total phenolics and flavonoids) in the produced callus. Stress biomarkers (hydrogen peroxide and malondialdehyde) were found in the control ranges except at 10 g/l DPS. The expression patterns of key genes involoved in secondary metabolism modulation, such as phenylalanine ammonia lyase (PAL), chalcone synthase (CHS), chalcone isomerase (CHI), flavonol synthase (FLS) and deoxyxylulose phosphate reductoisomerase (DXR), were triggered after DPS treatments. Moreover, the quantitative profiling of phenolic and flavonoid compounds showed that supplementation with DPS, especially at 8 g/l, led to pronounced increases in most of the measured compounds.ConclusionThe marked upregulation of eliciting-responsive genes and overproduction of secondary metabolites provide molecular-based evidence for intensifying the principal pathways of phenylpropanoid, flavonoid and terpenoid biosynthesis. Overall, the present in vitro study highlights the stimulating capacity of DPS utilization to improve the bioactive components of L. arabicus at the physiological and molecular levels, enhancing its potential as a medicinal herb.

An integrated transcriptome, metabolomic, and physiological investigation uncovered the underlying tolerance mechanisms of Monochoria korsakowii in response to acute/chronic cadmium exposure

Identifying the physiological response and tolerance mechanism of wetland plants to heavy metal exposure can provide theoretical guidance for an early warning for acute metal pollution and metal-contaminated water phytoremediation. A hydroponic experiment was employed to investigate variations in the antioxidant enzyme activity, chlorophyll content, and photosynthesis in leaves of Monochoria korsakowii under 0.12mM cadmium ion (Cd2+) acute (4d) and chronic (21d) exposure. Transcriptome and metabolome were analyzed to elucidate the underlying defensive strategies. The acute/chronic Cd2+ exposure decreased chlorophyll a and b contents, and disturbed photosynthesis in the leaves. The acute Cd2+ exposure increased catalase activity by 36.42%, while the chronic Cd2+ exposure markedly increased ascorbate peroxidase, superoxide dismutase, and glutathione peroxidase activities in the leaves. A total of 2 685 differentially expressed genes (DEGs) in the leaves were identified with the plants exposed to the acute/chronic Cd2+ contamination. In the acute Cd2+ exposure treatment, DEGs were preferentially enriched in the plant hormone transduction pathway, followed by phenylrpopanoid biosynthesis. However, the chronic Cd2+ exposure induced DEGs enriched in the biosynthesis of secondary metabolites pathway as priority. With acute/chronic Cd2+ exposure, a total of 157 and 227 differentially expressed metabolites were identified in the leaves. Conjoint transcriptome and metabolome analysis indicated the plant hormone signal transduction pathway and biosynthesis of secondary metabolites was preferentially activated by the acute and chronic Cd2+ exposure, respectively. The phenylpropanoid pathway functioned as a chemical defense, and the positive role of deoxyxylulose phosphate pathway in leaves against acute/chronic Cd2+ exposure was impaired.

Characterization of the enzymatic and multifunctional properties of Acinetobacter baumannii erythrose-4-phosphate dehydrogenase (E4PDH).

Infections due to Acinetobacter baumannii (A. baumannii) are rapidly increasing worldwide and consequently therapeutic options for treatment are limited. The emergence of multi drug resistant (MDR) strains has rendered available antibiotics ineffective, necessitating the urgent discovery of new drugs and drug targets. The vitamin B6 biosynthetic pathway has been considered as a potential antibacterial drug target but it is as yet uncharacterized for A. baumannii. In the current work, we have carried out in silico and biochemical characterization of Erythrose-4-phosphate dehydrogenase (E4PDH) (EC 1.2.1.72). This enzyme catalyzes the first step in the deoxyxylulose-5-phosphate (DXP) dependent Vitamin B6 biosynthetic pathway i.e. the conversion of d-erythrose-4-phosphate (E4P) to 4-Phosphoerythronate. E4PDH also possesses an additional activity whereby it can catalyze the conversion of Glyceraldehyde-3-phosphate (G3P) to 1,3 bisphosphoglycerate (1,3BPG). Our studies have revealed that this enzyme exhibits an alternate moonlighting function as a cell surface receptor for the human iron transport proteins transferrin (Tf) and lactoferrin (Lf). The present work reports the internalization of Tf and consequent iron acquisition as an alternate strategy for iron acquisition. Given its essential role in two crucial pathways i.e. metabolism and iron acquisition, A. baumannii E4PDH may play a vital role in bacterial pathogenesis.

MEP pathway products allosterically promote monomerization of deoxy-D-xylulose-5-phosphate synthase to feedback-regulate their supply

Isoprenoids are a very large and diverse family of metabolites required by all living organisms. All isoprenoids derive from the double-bond isomers isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), which are produced by the methylerythritol 4-phosphate (MEP) pathway in bacteria and plant plastids. It has been reported that IPP and DMAPP feedback-regulate the activity of deoxyxylulose 5-phosphate synthase (DXS), a dimeric enzyme that catalyzes the main flux-controlling step of the MEP pathway. Here we provide experimental insights into the underlying mechanism. Isothermal titration calorimetry and dynamic light scattering approaches showed that IPP and DMAPP can allosterically bind to DXS invitro, causing a size shift. In silico ligand binding site analysis and docking calculations identified a potential allosteric site in the contact region between the two monomers of the active DXS dimer. Modulation of IPP and DMAPP contents invivo followed by immunoblot analyses confirmed that high IPP/DMAPP levels resulted in monomerization and eventual aggregation of the enzyme in bacterial and plant cells. Loss of the enzymatically active dimeric conformation allows a fast and reversible reduction of DXS activity in response to a sudden increase or decrease in IPP/DMAPP supply, whereas aggregation and subsequent removal of monomers that would otherwise be available for dimerization appears to be a more drastic response in the case of persistent IPP/DMAPP overabundance (e.g., by a blockage in their conversion to downstream isoprenoids). Our results represent an important step toward understanding the regulation of the MEP pathway and rational design of biotechnological endeavors aimed at increasing isoprenoid contents in microbial and plant systems.

A direct enzymatic evaluation platform (DEEP) to fine-tuning pyridoxal 5'-phosphate-dependent proteins for cadaverine production.

Pyridoxal 5'-phosphate (pyridoxal phosphate, PLP) is an essential cofactor for multiple enzymatic reactions in industry. However, cofactor engineering based on PLP regeneration and related to the performance of enzymes in chemical production has rarely been discussed. First, we found that MG1655 strain was sensitive to nitrogen source and relied on different amino acids, thus the biomass was significantly reduced when PLP excess in the medium. Then, the six KEIO collection strains were applied to find out the prominent gene in deoxyxylulose-5-phosphate (DXP) pathway, where pdxB was superior in controlling cell growth. Therefore, the clustered regularly interspaced short palindromic repeats interference (CRISPRi) targeted on pdxB in MG1655 was employed to establish a novel direct enzymatic evaluation platform (DEEP) as a high-throughput tool and obtained the optimal modules for incorporating of PLP to enhance the biomass and activity of PLP-dependent enzymes simultaneously. As a result, the biomass has increased by 55% using PlacI promoter driven pyridoxine 5'-phosphate oxidase (PdxH) with a trace amount of precursor. When the strains incorporated DEEP and lysine decarboxylase (CadA), the cadaverine productivity was increased 32% due to the higher expression of CadA. DEEP is not only feasible for high-throughput screening of the best chassis for PLP engineering but also practical in fine-tuning the quantity and quality of enzymes.

Effects of herbivory on carotenoid biosynthesis and breakdown.

The carotenoid content of plants may be impacted by stress with major consequences for photosynthesis and photoprotection. Most carotenoid stress research, however, has concentrated on abiotic stresses, and we know little about how biological stresses, such as herbivory, alter profiles of plant carotenoids and their degradation products. For example, carotenoid derivatives such as β-cyclocitral and β-ionone have been recently shown to act as signals in plant growth and protection against oxidative stress and herbivory. To understand how carotenoid composition is influenced by herbivory, changes in biosynthesis and degradation should be investigated. This chapter describes methods to simulate herbivory in a simple reproducible fashion and to assess carotenoid biosynthesis and degradation. Carotenoid biosynthesis depends on precursors provided by the methylerythritol 4-phosphate (MEP) pathway, which converts pyruvate and glyceraldehyde-3-phosphate to the five-carbon units used for construction of larger isoprenoids. We present protocols to quantify the activity of the first enzyme of the MEP pathway, deoxy-xylulose 5-phosphate synthase (DXS), usually assumed to be rate-controlling, and to estimate the concentration of the first intermediate of the pathway, deoxy-xylulose 5-phosphate (DXP). We also discuss procedures to measure the formation of volatile carotenoid breakdown products after herbivory. To monitor the activity of carotenoid-specific biosynthetic enzymes, such as phytoene synthase, protocols are available elsewhere in this volume (Wurtzel, 2022b).

Metabolic Remodeling during Nitrogen Fixation in Zymomonas mobilis.

ABSTRACTZymomonas mobilis is an ethanologenic bacterium currently being developed for production of advanced biofuels. Recent studies have shown that Z. mobilis can fix dinitrogen gas (N2) as a sole nitrogen source. During N2 fixation, Z. mobilis exhibits increased biomass-specific rates of ethanol production. In order to better understand the physiology of Z. mobilis during N2 fixation and during changes in ammonium (NH4+) availability, we performed liquid chromatography-mass spectrometry (LC-MS)-based targeted metabolomics and shotgun proteomics under three regimes of nitrogen availability: continuous N2 fixation, gradual NH4+ depletion, and acute NH4+ addition to N2-fixing cells. We report dynamic changes in abundance of proteins and metabolites related to nitrogen fixation, motility, ammonium assimilation, amino acid biosynthesis, nucleotide biosynthesis, isoprenoid biosynthesis, and Entner-Doudoroff (ED) glycolysis, providing insight into the regulatory mechanisms that control these processes in Z. mobilis. Our analysis identified potential physiological mechanisms that may contribute to increased specific ethanol production during N2 fixation, including decreased activity of biosynthetic pathways, increased protein abundance of alcohol dehydrogenase (ADHI), and increased thermodynamic favorability of the ED pathway. Of particular relevance to advanced biofuel production, we found that intermediates in the methylerythritol phosphate (MEP) pathway for isoprenoid biosynthesis were depleted during N2 fixation, coinciding with decreased protein abundance of deoxyxylulose 5-phosphate synthase (DXS), the first enzyme in the pathway. This implies that DXS protein abundance serves as a native control point in regulating MEP pathway activity in Z. mobilis. The results of this study will inform metabolic engineering to further develop Z. mobilis as a platform organism for biofuel production.IMPORTANCE Biofuels and bioproducts have the potential to serve as environmentally sustainable replacements for petroleum-derived fuels and commodity molecules. Advanced fuels such as higher alcohols and isoprenoids are more suitable gasoline replacements than bioethanol. Developing microbial systems to generate advanced biofuels requires metabolic engineering to reroute carbon away from ethanol and other native products and toward desired pathways, such as the MEP pathway for isoprenoid biosynthesis. However, rational engineering of microbial metabolism relies on understanding metabolic control points, in terms of both enzyme activity and thermodynamic favorability. In Z. mobilis, the factors that control glycolytic rates, ethanol production, and isoprenoid production are still not fully understood. In this study, we performed metabolomic, proteomic, and thermodynamic analysis of Z. mobilis during N2 fixation. This analysis identified key changes in metabolite levels, enzyme abundance, and glycolytic thermodynamic favorability that occurred during changes in NH4+ availability, helping to inform future efforts in metabolic engineering.

Manipulation of DXP pathway for andrographolide production in callus cultures of Andrographis paniculata.

Greening of in vitro callus cultures and andrographolide over-accumulation was achieved by manipulating light exposure and media composition, when the biosynthetic cascade was channeled through the DXP pathway. Andrographolide, the primary biologically active compound of Andrographis paniculata, is produced through coordinated action of two pathways, the classical cytosolic mevalonate pathway and the alternative plastidial non-mevalonate pathway (Deoxy-xylulose Phosphate pathway). In vitro callus cultures of A. paniculata are useful sources of production, as well as, manipulation of andrographolide, and the present study was designed to explore the strategy of pathway inhibition for its overproduction. When the cytosolic mevalonate pathway blocker, lovastatin, was applied to callus cultures of A. paniculata, andrographolide production was enhanced in comparison to untreated control. In contrast, treatment of the callus tissue with the DXP-pathway blocker, fosmidomycin, led to depletion in andrographolide production. The present study also showed that silver nitrate, a potent elicitor of andrographolide production in in vitro callus culture, when added in combination with the pathway inhibitors resulted in alterations in andrographolide production. The highest andrographolide production was obtained in callus treated with a combination of silver nitrate and lovastatin, indicating a predominant role of the plastidial DXP pathway in andrographolide biosynthesis. A positive co-relation with chlorophyll content and andrographolide production in in vitro callus cultures (untreated and treated) observed also supported the above assumption. It could be inferred from this study that greening of callus tissue through organellar organization was a potent strategy for enhancing andrographolide accumulation in callus tissues of A. paniculata.

Knowns and Unknowns of Vitamin B6 Metabolism in Escherichia coli.

Vitamin B6 is an ensemble of six interconvertible vitamers: pyridoxine (PN), pyridoxamine (PM), pyridoxal (PL), and their 5'-phosphate derivatives, PNP, PMP, and PLP. Pyridoxal 5'-phosphate is a coenzyme in a variety of enzyme reactions concerning transformations of amino and amino acid compounds. This review summarizes all known and putative PLP-binding proteins found in the Escherichia coli MG1655 proteome. PLP can have toxic effects since it contains a very reactive aldehyde group at its 4' position that easily forms aldimines with primary and secondary amines and reacts with thiols. Most PLP is bound either to the enzymes that use it as a cofactor or to PLP carrier proteins, protected from the cellular environment but at the same time readily transferable to PLP-dependent apoenzymes. E. coli and its relatives synthesize PLP through the seven-step deoxyxylulose-5-phosphate (DXP)-dependent pathway. Other bacteria synthesize PLP in a single step, through a so-called DXP-independent pathway. Although the DXP-dependent pathway was the first to be revealed, the discovery of the widespread DXP-independent pathway determined a decline of interest in E. coli vitamin B6 metabolism. In E. coli, as in most organisms, PLP can also be obtained from PL, PN, and PM, imported from the environment or recycled from protein turnover, via a salvage pathway. Our review deals with all aspects of vitamin B6 metabolism in E. coli, from transcriptional to posttranslational regulation. A critical interpretation of results is presented, in particular, concerning the most obscure aspects of PLP homeostasis and delivery to PLP-dependent enzymes.

Integrated Transcriptomic and Metabolomic analysis reveals a transcriptional regulation network for the biosynthesis of carotenoids and flavonoids in \u2018Cara cara\u2019 navel Orange

BackgroundCarotenoids and flavonoids are important secondary metabolites in plants, which exert multiple bioactivities and benefits to human health. Although the genes that encode carotenogenesis and flavonoid biosynthetic enzymes are well characterized, the transcriptional regulatory mechanisms that are related to the pathway genes remain to be investigated. In this study, ‘Cara cara’ navel orange (CNO) fruit at four development stages were used to identify the key genes and TFs for carotenoids and flavonoids accumulation.ResultsIn this study, CNO was used to investigate the profiles of carotenoids and flavonoids by a combination of metabolomic and transcriptomic analyses. The important stage for the accumulation of the major carotenoid, lycopene was found to be at 120 days after florescence (DAF). The transcripts of five carotenogenesis genes were highly correlated with lycopene contents, and 16, 40, 48, 24 and 18 transcription factors (TFs) were predicted to potentially bind 1-deoxy-D-xylulose-5-phosphate synthase (DXS1), deoxyxylulose 5-phosphate reductoisomerase (DXR), geranylgeranyl diphosphate synthase (GGPPS2), phytoene synthase (PSY1) and lycopene β-cyclase (LCYB) promoters, respectively. Narirutin was the most abundant flavonoid in the flesh at the early stages, 60 DAF was the most important stage for the accumulation of flavonoids, and 17, 22, 14, 25, 24 and 16 TFs could potentially bind phenylalanine ammonia-lyase (PAL-1 and PAL-4), 4-Coumarate-CoA ligase (4CL-2 and 4CL-5), chalcone synthase (CHS-1) and chalcone isomerase (CHI) promoters, respectively. Furthermore, both sets of 15 candidate TFs might regulate at least three key genes and contribute to carotenoids/flavonoids accumulation in CNO fruit. Finally, a hierarchical model for the regulatory network among the pathway genes and TFs was proposed.ConclusionsCollectively, our results suggest that DXS1, DXR, GGPPS2, PSY1 and LCYB genes were the most important genes for carotenoids accumulation, while PAL-1, PAL-4, 4CL-2, 4CL-5, CHS-1 and CHI for flavonoids biosynthesis. A total of 24 TFs were postulated as co-regulators in both pathways directly, which might play important roles in carotenoids and flavonoids accumulation in CNO fruit.

Potent, specific MEPicides for treatment of zoonotic staphylococci.

Coagulase-positive staphylococci, which frequently colonize the mucosal surfaces of animals, also cause a spectrum of opportunistic infections including skin and soft tissue infections, urinary tract infections, pneumonia, and bacteremia. However, recent advances in bacterial identification have revealed that these common veterinary pathogens are in fact zoonoses that cause serious infections in human patients. The global spread of multidrug-resistant zoonotic staphylococci, in particular the emergence of methicillin-resistant organisms, is now a serious threat to both animal and human welfare. Accordingly, new therapeutic targets that can be exploited to combat staphylococcal infections are urgently needed. Enzymes of the methylerythritol phosphate pathway (MEP) of isoprenoid biosynthesis represent potential targets for treating zoonotic staphylococci. Here we demonstrate that fosmidomycin (FSM) inhibits the first step of the isoprenoid biosynthetic pathway catalyzed by deoxyxylulose phosphate reductoisomerase (DXR) in staphylococci. In addition, we have both enzymatically and structurally determined the mechanism by which FSM elicits its effect. Using a forward genetic screen, the glycerol-3-phosphate transporter GlpT that facilitates FSM uptake was identified in two zoonotic staphylococci, Staphylococcus schleiferi and Staphylococcus pseudintermedius. A series of lipophilic ester prodrugs (termed MEPicides) structurally related to FSM were synthesized, and data indicate that the presence of the prodrug moiety not only substantially increased potency of the inhibitors against staphylococci but also bypassed the need for GlpT-mediated cellular transport. Collectively, our data indicate that the prodrug MEPicides selectively and robustly inhibit DXR in zoonotic staphylococci, and further, that DXR represents a promising, druggable target for future development.

The organ-specific differential roles of rice DXS and DXR, the first two enzymes of the MEP pathway, in carotenoid metabolism in Oryza sativa leaves and seeds

BackgroundDeoxyxylulose 5-phosphate synthase (DXS) and deoxyxylulose 5-phosphate reductoisomerase (DXR) are the enzymes that catalyze the first two enzyme steps of the methylerythritol 4-phosphate (MEP) pathway to supply the isoprene building-blocks of carotenoids. Plant DXR and DXS enzymes have been reported to function differently depending on the plant species. In this study, the differential roles of rice DXS and DXR genes in carotenoid metabolism were investigated.ResultsThe accumulation of carotenoids in rice seeds co-expressing OsDXS2 and stPAC was largely enhanced by 3.4-fold relative to the stPAC seeds and 315.3-fold relative to non-transgenic (NT) seeds, while the overexpression of each OsDXS2 or OsDXR caused no positive effect on the accumulation of either carotenoids or chlorophylls in leaves and seeds, suggesting that OsDXS2 functions as a rate-limiting enzyme supplying IPP/DMAPPs to seed carotenoid metabolism, but OsDXR doesn’t in either leaves or seeds. The expressions of OsDXS1, OsPSY1, OsPSY2, and OsBCH2 genes were upregulated regardless of the reductions of chlorophylls and carotenoids in leaves; however, there was no significant change in the expression of most carotenogenic genes, even though there was a 315.3-fold increase in the amount of carotenoid in rice seeds. These non-proportional expression patterns in leaves and seeds suggest that those metabolic changes of carotenoids were associated with overexpression of the OsDXS2, OsDXR and stPAC transgenes, and the capacities of the intermediate biosynthetic enzymes might be much more important for those metabolic alterations than the transcript levels of intermediate biosynthetic genes are. Taken together, we propose a ‘Three Faucets and Cisterns Model’ about the relationship among the rate-limiting enzymes OsDXSs, OsPSYs, and OsBCHs as a “Faucet”, the biosynthetic capacity of intermediate metabolites as a “Cistern”, and the carotenoid accumulations as the content of “Cistern”.ConclusionOur study suggests that OsDXS2 plays an important role as a rate-limiting enzyme supplying IPP/DMAPPs to the seed-carotenoid accumulation, and rice seed carotenoid metabolism could be largely enhanced without any significant transcriptional alteration of carotenogenic genes. Finally, the “Three Faucets and Cisterns model” presents the extenuating circumstance to elucidate rice seed carotenoid metabolism.

Pyridoxal Reductase, PdxI, Is Critical for Salvage of Pyridoxal in Escherichia coli.

Pyridoxal 5'-phosphate (PLP) is the biologically active form of vitamin B6 and an essential cofactor in all organisms. In Escherichia coli, PLP is synthesized via the deoxyxylulose 5-phosphate (DXP)-dependent pathway that includes seven enzymatic steps and generates pyridoxine 5'-phosphate as an intermediate. Additionally, E. coli is able to salvage pyridoxal, pyridoxine, and pyridoxamine B6 vitamers to produce PLP using kinases PdxK/PdxY and pyridox(am)ine phosphate oxidase (PdxH). We found that E. coli strains blocked in PLP synthesis prior to the formation of pyridoxine 5'-phosphate (PNP) required significantly less exogenous pyridoxal (PL) than strains lacking pdxH and identified the conversion of PL to pyridoxine (PN) during cultivation to be the cause. Our data showed that PdxI, shown to have PL reductase activity in vitro, was required for the efficient salvage of PL in E. coli The pdxI+E. coli strains converted exogenous PL to PN during growth, while pdxI mutants did not. In total, the data herein demonstrated that PdxI is a critical enzyme in the salvage of PL by E. coliIMPORTANCE The biosynthetic pathway of pyridoxal 5'-phosphate (PLP) has extensively been studied in Escherichia coli, yet limited information is available about the vitamin B6 salvage pathway. We show that the protein PdxI (YdbC) is the primary pyridoxal (PL) reductase in E. coli and is involved in the salvage of PL. The orthologs of PdxI occur in a wide range of bacteria and plants, suggesting that PL reductase in the B6 salvage pathway is more widely distributed than previously expected.

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.

A two-step evolutionary process establishes a non-native vitamin B6 pathway in Bacillus subtilis.

Pyridoxal 5'-phosphate (PLP), the most important form of vitamin B6 serves as a cofactor for many proteins. Two alternative pathways for de novo PLP biosynthesis are known: the short deoxy-xylulose-5-phosphate (DXP)-independent pathway, which is present in the Gram-positive model bacterium Bacillus subtilis and the longer DXP-dependent pathway, which has been intensively studied in the Gram-negative model bacterium Escherichia coli. Previous studies revealed that bacteria contain many promiscuous enzymes causing a so-called 'underground metabolism', which can be important for the evolution of novel pathways. Here, we evaluated the potential of B. subtilis to use a truncated non-native DXP-dependent PLP pathway from E. coli for PLP synthesis. Adaptive laboratory evolution experiments revealed that two non-native enzymes catalysing the last steps of the DXP-dependent PLP pathway and two genomic alterations are sufficient to allow growth of vitamin B6 auxotrophic bacteria as rapid as the wild type. Thus, the existence of an underground metabolism in B. subtilis facilitates the generation of a pathway for synthesis of PLP using parts of a non-native vitamin B6 pathway. The introduction of non-native enzymes into a metabolic network and rewiring of native metabolism could be helpful to generate pathways that might be optimized for producing valuable substances.

MEPicides: potent antimalarial prodrugs targeting isoprenoid biosynthesis

The emergence of Plasmodium falciparum resistant to frontline therapeutics has prompted efforts to identify and validate agents with novel mechanisms of action. MEPicides represent a new class of antimalarials that inhibit enzymes of the methylerythritol phosphate (MEP) pathway of isoprenoid biosynthesis, including the clinically validated target, deoxyxylulose phosphate reductoisomerase (Dxr). Here we describe RCB-185, a lipophilic prodrug with nanomolar activity against asexual parasites. Growth of P. falciparum treated with RCB-185 was rescued by isoprenoid precursor supplementation, and treatment substantially reduced metabolite levels downstream of the Dxr enzyme. In addition, parasites that produced higher levels of the Dxr substrate were resistant to RCB-185. Notably, environmental isolates resistant to current therapies remained sensitive to RCB-185, the compound effectively treated sexually-committed parasites, and was both safe and efficacious in malaria-infected mice. Collectively, our data demonstrate that RCB-185 potently and selectively inhibits Dxr in P. falciparum, and represents a promising lead compound for further drug development.

Genetic Diversity and Association Study of Aromatics in Grapevine

Monoterpenoid metabolism and aroma compounds are influenced by genetic characteristics. Linalool, α-terpineol, nerol, and geraniol are primary monoterpenoids that have previously been studied in grape (Vitis vinifera) berries. Previous studies were restricted by the lack of relevant studies investigating population structure and the regulatory mechanism underlying monoterpenoid synthesis. In this study, a total of 1133 alleles were amplified, with each locus having on average 6.06 alleles. We also assessed the genetic variability among the genotypes based on 187 microsatellite primer pairs amplified in 96 grape genotypes. The results of the phylogenetic tree analysis showed that the grapevine accessions grouped into five genetic clusters that largely coincided with the recognized species classification and the result of principal coordinates analysis (PCoA). The molecular characterization of these accessions provides insight into genetic diversity, population structure, and linkage disequilibrium (LD) in grapevines. A total of 51 quantitative trait loci (QTLs) were detected that were significantly associated with linalool, α-terpineol, nerol, and geraniol. We found that Deoxyxylulose phosphate synthase (DXS) was located in the region UDV060 on linkage group (LG) 5, whereas Farnesyl diphosphate synthase (FPPS) and Hydroxymethylbutenyl diphosphate reductase (HDR) were located in the VLG19-I-1 and VLG3-A-1 regions, respectively. These novel QTLs will potentially assist in the screening of aroma compounds in grapevines.