Pyruvate lies at a pivotal node of carbon metabolism in eukaryotes. It is involved in diverse metabolic pathways in multiple organelles, and its interorganelle shuttling is crucial for cell fitness. Many apicomplexan parasites harbor a unique organelle called the apicoplast that houses metabolic pathways like fatty acid and isoprenoid precursor biosyntheses, requiring pyruvate as a substrate. However, how pyruvate is supplied in the apicoplast remains enigmatic. Here, deploying the zoonotic parasite Toxoplasma gondii as a model apicomplexan, we identified two proteins residing in the apicoplast membranes that together constitute a functional apicoplast pyruvate carrier (APC) to mediate the import of cytosolic pyruvate. Depletion of APC results in reduced activities of metabolic pathways in the apicoplast and impaired integrity of this organelle, leading to parasite growth arrest. APC is a pyruvate transporter in diverse apicomplexan parasites, suggesting a common strategy for pyruvate acquisition by the apicoplast in these clinically relevant intracellular pathogens.

Volatile organic compounds (VOCs) emitted by plants may help in understanding the status of a plant\'s physiology and its coping with mild to severe stress. Future climatic projections reveal that shifts in temperature and CO2 availability will occur, and plants may incur the uncoupling of carbon assimilation and synthesis of key molecules. This study explores the patterns of emissions of key VOCs (isoprene, methanol, acetaldehyde, and acetic acid) emitted by poplar leaves (more than 350) under a combined gradient of temperature (12-42 °C) and air CO2 concentration (400-1500 ppm), along with measurements of photosynthetic rates and stomatal conductance. Isoprene emission exhibited a rise with temperature and CO2 availability, peaking at 39 °C, the temperature at which methanol emission started to peak, illustrating the limit of stress tolerance to severe damage. Isoprene emission was uncoupled from the photosynthesis rate, indicating a shift from the carbon source for isoprene synthesis, while assimilation was decreased. Methanol and acetaldehyde emissions were correlated with stomatal conductance and peaked at 25 °C and 1200 ppm CO2. Acetic acid emissions lacked a clear correlation with stomatal conductance and the emission pattern of its precursor acetaldehyde. This study offers crucial insights into the limitations of photosynthetic carbon and stress tolerance.

In plants, the plastidial mevalonate (MVA)-independent pathway is required for the modification with geranylgeranyl groups of CaaL-motif proteins, which are substrates of protein geranylgeranyltransferase type-I (PGGT-I). As a consequence, fosmidomycin, a specific inhibitor of 1-deoxy-d-xylulose (DX)-5 phosphate reductoisomerase/DXR, the second enzyme in this so-called methylerythritol phosphate (MEP) pathway, also acts as an effective inhibitor of protein prenylation. This can be visualized in plant cells by confocal microscopy by expressing GFP-CaM-CVIL, a prenylation sensor protein. After treatment with fosmidomycin, the plasma membrane localization of this GFP-based sensor is altered, and a nuclear distribution of fluorescence is observed instead. In tobacco cells, a visual screen of conditions allowing membrane localization in the presence of fosmidomycin identified jasmonic acid methyl esther (MeJA) as a chemical capable of gradually overcoming inhibition. Using Arabidopsis protein prenyltransferase loss-of-function mutant lines expressing GFP-CaM-CVIL proteins, we demonstrated that in the presence of MeJA, protein farnesyltransferase (PFT) can modify the GFP-CaM-CVIL sensor, a substrate the enzyme does not recognize under standard conditions. Similar to MeJA, farnesol and MVA also alter the protein substrate specificity of PFT, whereas DX and geranylgeraniol have limited or no effect. Our data suggest that MeJA adjusts the protein substrate specificity of PFT by promoting a metabolic cross-talk directing the origin of the prenyl group used to modify the protein. MVA, or an MVA-derived metabolite, appears to be a key metabolic intermediate for this change in substrate specificity.

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.

In a vast majority of bacteria, protozoa and plants, the methylerythritol phosphate (MEP) pathway is utilized for the synthesis of isopentenyl diphosphate (IDP) and dimethylallyl diphosphate (DMADP), which are precursors for isoprenoids. Isoprenoids, such as cholesterol and coenzyme Q, play a variety of crucial roles in physiological activities, including cell-membrane formation, protein degradation, cell apoptosis, and transcription regulation. In contrast, humans employ the mevalonate (MVA) pathway for the production of IDP and DMADP, rendering proteins in the MEP pathway appealing targets for antimicrobial agents. This pathway consists of seven consecutive enzymatic reactions, of which 4-diphosphocytidyl-2C-methyl-D-erythritol synthase (IspD) and 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (IspF) catalyze the third and fifth steps, respectively. In this study, we characterized the enzymatic activities and protein structures of Helicobacter pylori IspDF and Acinetobacter baumannii IspD. Then, using the direct interaction-based thermal shift assay, we conducted a compound screening of an approved drug library and identified 27 hit compounds potentially binding to AbIspD. Among them, two natural products, rosmarinic acid and tanshinone IIA sodium sulfonate, exhibited inhibitory activities against HpIspDF and AbIspD, by competing with one of the substrates, MEP. Moreover, tanshinone IIA sodium sulfonate also demonstrated certain antibacterial effects against H. pylori. In summary, we identified two IspD inhibitors from approved ingredients, broadening the scope for antibiotic discovery targeting the MEP pathway.

In Arabidopsis, the plastidial isoform of phosphoglucose isomerase, PGI1, mediates growth and photosynthesis, likely due to its involvement in the vascular production of cytokinins (CK). To examine this hypothesis, we characterized pgi1-2 knockout plants impaired in PGI1 and pgi1-2 plants specifically expressing PGI1 in root tips and vascular tissues. Moreover, to investigate whether the phenotype of pgi1-2 plants is due to impairments in the plastidial oxidative pentose phosphate pathway (OPPP) or the glycolytic pathway, we characterized pgl3-1 plants with reduced OPPP and pfk4pfk5 knockout plants impaired in plastidial glycolysis. Compared with wild-type (WT) leaves, pgi1-2 leaves exhibited weaker expression of photosynthesis- and 2-C-methyl-D-erythritol 4-P (MEP) pathway-related proteins, and stronger expression of oxidative stress protection-related enzymes. Consistently, pgi1-2 leaves accumulated lower levels of chlorophyll, and higher levels of tocopherols, flavonols and anthocyanins than the WT. Vascular- and root tip-specific PGI1 expression countered the reduced photosynthesis, low MEP pathway-derived CK content, dwarf phenotype and the metabolic characteristics of pgi1-2 plants, reverting them to WT-like levels. Moreover, pgl3-1, but not pfk4pfk5 plants phenocopied pgi1-2. Histochemical analyses of plants expressing GUS under the control of promoter regions of genes encoding plastidial OPPP enzymes exhibited strong GUS activity in root tips and vascular tissues. Overall, our findings show that root tip and vascular PGI1-mediated plastidial OPPP activity affects photosynthesis and growth through mechanisms involving long-distance modulation of the leaf proteome by MEP pathway-derived CKs.

Fragrance is an important feature of ornamental lilies. Components of volatile substances and important genes for monoterpene synthesis in the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway were examined in this study. Twenty volatile compounds (2 in the budding stage, 3 in the initial flowering stage, 7 in the semi-flowering stage, 17 in the full-flowering stage, and 5 in withering stage) were detected in the Oriental lily \'Sorbonne\' using gas chromatography-mass spectrometry. The semi- and full-flowering stages were key periods for volatile substance production and enzyme function. Sequence assembly from samples collected during all flowering stages resulted in the detection of 274,849 genes and 129,017 transcripts. RNA sequencing and heatmapping led to the detection of genes in the MEP monoterpene metabolism pathway. Through gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway analysis, we extracted key genes (LiDXS2, LiLIS, and LiMYS) and transcription factors (in the bHLH, MYB, HD-ZIP, and NAC families) associated with the MEP pathway. Tissue localization revealed that LiDXS2, LiLIS, and LiMYS were expressed in Lilium \'Sorbonne\' petals in the full-flowering stage. Genes regulating the 1-deoxy-D-X-lignone-5-phosphate synthase family of rate-limiting enzymes, involved in the first step of monoterpene synthesis, showed high expression in the semi- and full-flowering stages. LiDXS2 was cloned and localized in chloroplast subcells. The relative expression of terpene-related genes in the MEP and mevalonic acid pathways of wild-type and LiLIS/LiMYS transgenic Arabidopsis thaliana, and changes in chemical composition, confirmed that LiLIS/LiMYS regulates the monoterpene synthesis pathway. The results of this study provide a theoretical basis for the synthesis of lily aromatic substances and the cultivation of new garden flower varieties.

In this study, we identified three novel compound classes with potent activity against Plasmodium falciparum, the most dangerous human malarial parasite. Resistance of this pathogen to known drugs is increasing, and compounds with different modes of action are urgently needed. One promising drug target is the enzyme 1-deoxy-d-xylulose-5-phosphate synthase (DXPS) of the methylerythritol 4-phosphate (MEP) pathway for which we have previously identified three active compound classes against Mycobacterium tuberculosis. The close structural similarities of the active sites of the DXPS enzymes of P. falciparum and M. tuberculosis prompted investigation of their antiparasitic action, all classes display good cell-based activity. Through structure-activity relationship studies, we increased their antimalarial potency and two classes also show good metabolic stability and low toxicity against human liver cells. The most active compound 1 inhibits the growth of blood-stage P. falciparum with an IC50 of 600 nM. The results from three different methods for target validation of compound 1 suggest no engagement of DXPS. All inhibitor classes are active against chloroquine-resistant strains, confirming a new mode of action that has to be further investigated.

The emergence of carbapenem-resistant Acinetobacter baumannii, a highly concerning bacterial species designated as a Priority 1: Critical pathogen by the WHO, has become a formidable global threat. In this study, we utilised computational methods to explore the potent molecules capable of inhibiting the IspC enzyme, which plays a crucial role in the methylerythritol 4-phosphate (MEP) biosynthetic pathway. Employing high-throughput virtual screening of small molecules from the Enamine library, we focused on the highly conserved substrate binding site of the DXR target protein, resulting in the identification of 1000 potential compounds. Among these compounds, we selected the top two candidates (Z2615855584 and Z2206320703) based on Lipinski\'s rule of Five and ADMET filters, along with FR900098, a known IspC inhibitor, and DXP, the substrate of IspC, for molecular dynamics (MD) simulations. The MD simulation trajectories revealed remarkable structural and thermodynamic stability, as well as strong binding affinity, for all the IspC-ligand complexes. Furthermore, binding free energy calculations based on MM/PBSA (Molecular Mechanics/Poisson-Boltzmann Surface Area) methodology demonstrated significant interactions between the selected ligand molecules and IspC. Taking into consideration all the aforementioned criteria, we suggest Z2206320703 as the potent lead candidate against IspC.
UNASSIGNED: The online version contains supplementary material available at 10.1007/s13205-024-03923-w.

Cyanobacteria are the prospective biosolar cell factories to produce a range of bioproducts through CO2 sequestration. Farnesene is a sesquiterpene with an array of applications in biofuels, pest management, cosmetics, flavours and fragrances. This is the first time a codon-optimized farnesene synthase (AFS) gene is engineered into the genomic neutral site of Synechococcus elongatus UTEX 2973 for farnesene synthesis through its endogenous methylerythritol phosphate (MEP) pathway, rendering UTEX AFS strain. Similarly, bottleneck gene(s) of the MEP pathway, 1-deoxy-D-xylulose-5-phosphate synthase (dxs) and/or fusion of isopentenyl diphosphate isomerase and farnesyl diphosphate synthase (idispA) were engineered engendering UTEX AFS::dxs, UTEX AFS::idispA and UTEX AFS::dxs::idispA strains. UTEX AFS::dxs::idispA achieves farnesene productivity of 2.57 mg/L/day, the highest among engineered cyanobacterial strains studied so far. It demonstrates farnesene production, which is 31.3-times higher than the UTEX AFS strain. Moreover, the engineered strains show similar productivity over a three-month period, stipulating the genetic stability of the strains.