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1
Banerjee, A., and T. D. Sharkey. "Methylerythritol 4-phosphate (MEP) pathway metabolic regulation." Nat. Prod. Rep. 31, no. 8 (2014): 1043–55. http://dx.doi.org/10.1039/c3np70124g.
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The methylerythritol 4-phosphate pathway provides precursors for isoprenoids in bacteria, some eukaryotic parasites, and chloroplasts of plants. Metabolic regulatory mechanisms control flux through the pathway and the concentration of a central intermediate, methylerythritol cyclodiphosphate.
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Testa, Charles A., and L. Jeffrey Johnson. "A Whole-Cell Phenotypic Screening Platform for Identifying Methylerythritol Phosphate Pathway-Selective Inhibitors as Novel Antibacterial Agents." Antimicrobial Agents and Chemotherapy 56, no. 9 (July 9, 2012): 4906–13. http://dx.doi.org/10.1128/aac.00987-12.
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ABSTRACTIsoprenoid biosynthesis is essential for survival of all living organisms. More than 50,000 unique isoprenoids occur naturally, with each constructed from two simple five-carbon precursors: isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Two pathways for the biosynthesis of IPP and DMAPP are found in nature. Humans exclusively use the mevalonate (MVA) pathway, while most bacteria, including all Gram-negative and many Gram-positive species, use the unrelated methylerythritol phosphate (MEP) pathway. Here we report the development of a novel, whole-cell phenotypic screening platform to identify compounds that selectively inhibit the MEP pathway. Strains ofSalmonella entericaserovar Typhimurium were engineered to have separately inducible MEP (native) and MVA (nonnative) pathways. These strains, RMC26 and CT31-7d, were then used to differentiate MVA pathway- and MEP pathway-specific perturbation. Compounds that inhibit MEP pathway-dependent bacterial growth but leave MVA-dependent growth unaffected represent MEP pathway-selective antibacterials. This screening platform offers three significant results. First, the compound is antibacterial and is therefore cell permeant, enabling access to the intracellular target. Second, the compound inhibits one or more MEP pathway enzymes. Third, the MVA pathway is unaffected, suggesting selectivity for targeting the bacterial versus host pathway. The cell lines also display increased sensitivity to two reported MEP pathway-specific inhibitors, further biasing the platform toward inhibitors selective for the MEP pathway. We demonstrate development of a robust, high-throughput screening platform that combines phenotypic and target-based screening that can identify MEP pathway-selective antibacterials simply by monitoring optical density as the readout for cell growth/inhibition.
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Cassera, María B., Fabio C. Gozzo, Fabio L. D'Alexandri, Emilio F. Merino, Hernando A. del Portillo, Valnice J. Peres, Igor C. Almeida, et al. "The Methylerythritol Phosphate Pathway Is Functionally Active in All Intraerythrocytic Stages ofPlasmodium falciparum." Journal of Biological Chemistry 279, no. 50 (September 27, 2004): 51749–59. http://dx.doi.org/10.1074/jbc.m408360200.
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Two genes encoding the enzymes 1-deoxy-d-xylulose-5-phosphate synthase and 1-deoxy-d-xylulose-5-phosphate reductoisomerase have been recently identified, suggesting that isoprenoid biosynthesis inPlasmodium falciparumdepends on the methylerythritol phosphate (MEP) pathway, and that fosmidomycin could inhibit the activity of 1-deoxy-d-xylulose-5-phosphate reductoisomerase. The metabolite 1-deoxy-d-xylulose-5-phosphate is not only an intermediate of the MEP pathway for the biosynthesis of isopentenyl diphosphate but is also involved in the biosynthesis of thiamin (vitamin B1) and pyridoxal (vitamin B6) in plants and many microorganisms. Herein we report the first isolation and characterization of most downstream intermediates of the MEP pathway in the three intraerythrocytic stages ofP. falciparum. These include, 1-deoxy-d-xylulose-5-phosphate, 2-C-methyl-d-erythritol-4-phosphate, 4-(cytidine-5-diphospho)-2-C-methyl-d-erythritol, 4-(cytidine-5-diphospho)-2-C-methyl-d-erythritol-2-phosphate, and 2-C-methyl-d-erythritol-2,4-cyclodiphosphate. These intermediates were purified by HPLC and structurally characterized via biochemical and electrospray mass spectrometric analyses. We have also investigated the effect of fosmidomycin on the biosynthesis of each intermediate of this pathway and isoprenoid biosynthesis (dolichols and ubiquinones). For the first time, therefore, it is demonstrated that the MEP pathway is functionally active in all intraerythrocytic forms ofP. falciparum, andde novobiosynthesis of pyridoxal in a protozoan is reported. Its absence in the human host makes both pathways very attractive as potential new targets for antimalarial drug development.
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Chen, Lijia, Hui Tong, Mingxuan Wang, Jianhua Zhu, Jiachen Zi, Liyan Song, and Rongmin Yu. "Effect of Enzyme Inhibitors on Terpene Trilactones Biosynthesis and Gene Expression Profiling in Ginkgo biloba Cultured Cells." Natural Product Communications 10, no. 12 (December 2015): 1934578X1501001. http://dx.doi.org/10.1177/1934578x1501001205.
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The biosynthetic pathway of terpene trilactones of Ginkgo biloba is unclear. In this present study, suspension cultured cells of G. biloba were used to explore the regulation of the mevalonic acid (MVA) and methylerythritol 4-phosphate (MEP) pathways in response to specific enzyme inhibitors (lovastatin and clomazone). The results showed that the biosynthesis of bilobalide was more highly correlated with the MVA pathway, and the biosynthesis of ginkgolides was more highly correlated with the MEP pathway. Meanwhile, according to the results, it could be speculated that bilobalide might be a product of ginkgolide metabolism.
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Zeidler, J., J. Schwender, C. Mueller, and H. K. Lichtenthaler. "The non-mevalonate isoprenoid biosynthesis of plants as a test system for drugs against malaria and pathogenic bacteria." Biochemical Society Transactions 28, no. 6 (December 1, 2000): 796–98. http://dx.doi.org/10.1042/bst0280796.
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Two plant test systems are presented in the search for new inhibitors of the non-mevalonate isoprenoid pathway. A derivative of clomazone appears to be an inhibitor of the deoxyxylulose 5-phosphate/methylerythritol 4-phosphate (DOXP/MEP) pathway of isoprenoid formation.
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Kadian, Kavita, Yash Gupta, Harsh Vardhan Singh, Prakasha Kempaiah, and Manmeet Rawat. "Apicoplast Metabolism: Parasite’s Achilles’ Heel." Current Topics in Medicinal Chemistry 18, no. 22 (January 10, 2019): 1987–97. http://dx.doi.org/10.2174/1568026619666181130134742.
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Malaria continues to impinge heavily on mankind, with five continents still under its clasp. Widespread and rapid emergence of drug resistance in the Plasmodium parasite to current therapies accentuate the quest for novel drug targets and antimalarial compounds. Plasmodium parasites, maintain a non-photosynthetic relict organelle known as Apicoplast. Among the four major pathways of Apicoplast, biosynthesis of isoprenoids via Methylerythritol phosphate (MEP) pathway is the only indispensable function of Apicoplast that occurs during different stages of the malaria parasite. Moreover, the human host lacks MEP pathway. MEP pathway is a validated repertoire of novel antimalarial and antibacterial drug targets. Fosmidomycin, an efficacious antimalarial compound against IspC enzyme of MEP pathway is already in clinical trials as a combination drugs. Exploitation of other enzymes of MEP pathway would provide a much-needed impetus to the antimalarial drug discovery programs for the elimination of malaria. We outline the cardinal features of the MEP pathway enzymes and progress made towards the characterization of new inhibitors.
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Cornish, Rita M., John R. Roth, and C. Dale Poulter. "Lethal Mutations in the Isoprenoid Pathway of Salmonella enterica." Journal of Bacteriology 188, no. 4 (February 15, 2006): 1444–50. http://dx.doi.org/10.1128/jb.188.4.1444-1450.2006.
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ABSTRACT Essential isoprenoid compounds are synthesized using the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway in many gram-negative bacteria, some gram-positive bacteria, some apicomplexan parasites, and plant chloroplasts. The alternative mevalonate pathway is found in archaea and eukaryotes, including cytosolic biosynthesis in plants. The existence of orthogonal essential pathways in eukaryotes and bacteria makes the MEP pathway an attractive target for the development of antimicrobial agents. A system is described for identifying mutations in the MEP pathway of Salmonella enterica serovar Typhimurium. Using this system, point mutations induced by diethyl sulfate were found in the all genes of the essential MEP pathway and also in genes involved in uptake of methylerythritol. Curiously, none of the MEP pathway genes could be identified in the same parent strain by transposon mutagenesis, despite extensive searches. The results complement the biochemical and bioinformatic approaches to the elucidation of the genes involved in the MEP pathway and also identify key residues for activity in the enzymes of the pathway.
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Pérez-Gil, Jordi, and Manuel Rodríguez-Concepción. "Metabolic plasticity for isoprenoid biosynthesis in bacteria." Biochemical Journal 452, no. 1 (April 25, 2013): 19–25. http://dx.doi.org/10.1042/bj20121899.
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Isoprenoids are a large family of compounds synthesized by all free-living organisms. In most bacteria, the common precursors of all isoprenoids are produced by the MEP (methylerythritol 4-phosphate) pathway. The MEP pathway is absent from archaea, fungi and animals (including humans), which synthesize their isoprenoid precursors using the completely unrelated MVA (mevalonate) pathway. Because the MEP pathway is essential in most bacterial pathogens (as well as in the malaria parasites), it has been proposed as a promising new target for the development of novel anti-infective agents. However, bacteria show a remarkable plasticity for isoprenoid biosynthesis that should be taken into account when targeting this metabolic pathway for the development of new antibiotics. For example, a few bacteria use the MVA pathway instead of the MEP pathway, whereas others possess the two full pathways, and some parasitic strains lack both the MVA and the MEP pathways (probably because they obtain their isoprenoids from host cells). Moreover, alternative enzymes and metabolic intermediates to those of the canonical MVA or MEP pathways exist in some organisms. Recent work has also shown that resistance to a block of the first steps of the MEP pathway can easily be developed because several enzymes unrelated to isoprenoid biosynthesis can produce pathway intermediates upon spontaneous mutations. In the present review, we discuss the major advances in our knowledge of the biochemical toolbox exploited by bacteria to synthesize the universal precursors for their essential isoprenoids.
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Banerjee, Aparajita, Yan Wu, Rahul Banerjee, Yue Li, Honggao Yan, and Thomas D. Sharkey. "Feedback Inhibition of Deoxy-d-xylulose-5-phosphate Synthase Regulates the Methylerythritol 4-Phosphate Pathway." Journal of Biological Chemistry 288, no. 23 (April 23, 2013): 16926–36. http://dx.doi.org/10.1074/jbc.m113.464636.
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The 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway leads to the biosynthesis of isopentenyl diphosphate (IDP) and dimethylallyl diphosphate (DMADP), the precursors for isoprene and higher isoprenoids. Isoprene has significant effects on atmospheric chemistry, whereas other isoprenoids have diverse roles ranging from various biological processes to applications in commercial uses. Understanding the metabolic regulation of the MEP pathway is important considering the numerous applications of this pathway. The 1-deoxy-d-xylulose-5-phosphate synthase (DXS) enzyme was cloned from Populus trichocarpa, and the recombinant protein (PtDXS) was purified from Escherichia coli. The steady-state kinetic parameters were measured by a coupled enzyme assay. An LC-MS/MS-based assay involving the direct quantification of the end product of the enzymatic reaction, 1-deoxy-d-xylulose 5-phosphate (DXP), was developed. The effect of different metabolites of the MEP pathway on PtDXS activity was tested. PtDXS was inhibited by IDP and DMADP. Both of these metabolites compete with thiamine pyrophosphate for binding with the enzyme. An atomic structural model of PtDXS in complex with thiamine pyrophosphate and Mg2+ was built by homology modeling and refined by molecular dynamics simulations. The refined structure was used to model the binding of IDP and DMADP and indicated that IDP and DMADP might bind with the enzyme in a manner very similar to the binding of thiamine pyrophosphate. The feedback inhibition of PtDXS by IDP and DMADP constitutes an important mechanism of metabolic regulation of the MEP pathway and indicates that thiamine pyrophosphate-dependent enzymes may often be affected by IDP and DMADP.
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Killiny, Nabil. "Silencing Phytoene Desaturase Causes Alteration in Monoterpene Volatiles Belonging to the Methylerythritol Phosphate Pathway." Plants 11, no. 3 (January 20, 2022): 276. http://dx.doi.org/10.3390/plants11030276.
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Volatile organic compounds (VOCs) are a large group of lipophilic hydrocarbon compounds derived from different biosynthetic pathways in plants. VOCs are produced and released from plants as a defense mechanism against biotic and abiotic stresses. They are involved in communication with the surrounding environment including plant-to-plant interactions and attracting or repelling insects. In citrus, phytoene desaturase (PDS), a precursor of the carotenoid biosynthetic pathway has been silenced using the Citrus tristeza virus-induced gene silencing technique. Silencing PDS resulted in a reduction of carotenoid contents and in the photobleaching phenotype in leaves. Interestingly, the strength of the phenotype was varied within the plants due to the unequal distribution of virus particles. Using solid-phase microextraction (SPME), fibers released VOCs from leaves with gradient degrees of the photobleaching phenotype were collected and analyzed in gas chromatography-mass spectrophotometry (GC-MS). Overall, 47 VOCs belonging to 12 chemically distinguished groups were detected and identified using authentic standards. Simple linear regression showed that monoterpenes belonging to methylerythritol phosphate (MEP) were significantly corrected with the degrees of photobleaching (carotenoid content). Both carotenoids and MEP biosynthetic pathways occurred in the plastid. Thus, we provide preliminary evidence for a potential role of carotenoids in supporting the MEP pathway and/or the production of monoterpenes.
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Choi, Seoung-Ryoung, and Prabagaran Narayanasamy. "Investigating Novel IspE Inhibitors of the MEP Pathway in Mycobacterium." Microorganisms 12, no. 1 (December 21, 2023): 18. http://dx.doi.org/10.3390/microorganisms12010018.
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In a recent effort to mitigate harm from human pathogens, many biosynthetic pathways have been extensively evaluated for their ability to inhibit pathogen growth and to determine drug targets. One of the important products/targets of such pathways is isopentenyl diphosphate. Isopentenyl diphosphate is the universal precursor of isoprenoids, which are essential for the normal functioning of microorganisms. In general, two biosynthetic pathways lead to the formation of isopentenyl diphosphate: (1) the mevalonate pathway in animals; and (2) the non-mevalonate or methylerythritol phosphate (MEP) in many bacteria, and some protozoa and plants. Because the MEP pathway is not found in mammalian cells, it is considered an attractive target for the development of antimicrobials against a variety of human pathogens, including Mycobacterium tuberculosis (M.tb). In the MEP pathway, 4-diphosphocytidyl-2-c-methyl-d-erythritol kinase (IspE) phosphorylates 4-diphosphocytidyl-2-C-methyl-D-erythritol (CDPME) to form 4-diphosphocytidyl-2-C-methyl-D-erythritol 2-phosphate (CDPME2P). A virtual high-throughput screening against 15 million compounds was carried out by docking IspE protein. We identified an active heterotricyclic compound which showed enzymatic activity; namely, IC50 of 6 µg/mL against M.tb IspE and a MIC of 12 µg/mL against M.tb (H37Rv). Hence, we designed and synthesized similar new heterotricyclic compounds and tested them against mycobacterium, observing a MIC of 5 µg/mL against M. avium. This study will provide the critical insight necessary for developing novel antimicrobials that target the MEP pathways in pathogens.
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Rohmer, M. "Mevalonate-independent methylerythritol phosphate pathway for isoprenoid biosynthesis. Elucidation and distribution." Pure and Applied Chemistry 75, no. 2-3 (January 1, 2003): 375–88. http://dx.doi.org/10.1351/pac200375020375.
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A long-overlooked metabolic pathway for isoprenoid biosynthesis, the mevalonate-independent methylerythritol phosphate (MEP) pathway, is present in many bacteria and in the chloroplasts of all phototrophic organisms. It represents an alternative to the well known mevalonate pathway, which is present in animals, fungi, plant cytoplasm, archaebacteria, and some eubacteria. This contribution summarizes key steps of its elucidation and the state-of-the-art knowledge of this biosynthetic pathway, which represents a novel target for antibacterial and antiparasitic drugs.
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González-Cabanelas, Diego, Erica Perreca, Johann M. Rohwer, Axel Schmidt, Tobias Engl, Bettina Raguschke, Jonathan Gershenzon, and Louwrance P. Wright. "Deoxyxylulose 5-Phosphate Synthase Does Not Play a Major Role in Regulating the Methylerythritol 4-Phosphate Pathway in Poplar." International Journal of Molecular Sciences 25, no. 8 (April 10, 2024): 4181. http://dx.doi.org/10.3390/ijms25084181.
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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.
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Zhao, Yaru, Jianming Yang, Bo Qin, Yonghao Li, Yuanzhang Sun, Sizheng Su, and Mo Xian. "Biosynthesis of isoprene in Escherichia coli via methylerythritol phosphate (MEP) pathway." Applied Microbiology and Biotechnology 90, no. 6 (April 6, 2011): 1915–22. http://dx.doi.org/10.1007/s00253-011-3199-1.
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Henry, Laura K., Michael Gutensohn, Suzanne T. Thomas, Joseph P. Noel, and Natalia Dudareva. "Orthologs of the archaeal isopentenyl phosphate kinase regulate terpenoid production in plants." Proceedings of the National Academy of Sciences 112, no. 32 (July 27, 2015): 10050–55. http://dx.doi.org/10.1073/pnas.1504798112.
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Terpenoids, compounds found in all domains of life, represent the largest class of natural products with essential roles in their hosts. All terpenoids originate from the five-carbon building blocks, isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP), which can be derived from the mevalonic acid (MVA) and methylerythritol phosphate (MEP) pathways. The absence of two components of the MVA pathway from archaeal genomes led to the discovery of an alternative MVA pathway with isopentenyl phosphate kinase (IPK) catalyzing the final step, the formation of IPP. Despite the fact that plants contain the complete classical MVA pathway, IPK homologs were identified in every sequenced green plant genome. Here, we show that IPK is indeed a member of the plant terpenoid metabolic network. It is localized in the cytosol and is coexpressed with MVA pathway and downstream terpenoid network genes. In planta, IPK acts in parallel with the MVA pathway and plays an important role in regulating the formation of both MVA and MEP pathway-derived terpenoid compounds by controlling the ratio of IP/DMAP to IPP/DMAPP. IP and DMAP can also competitively inhibit farnesyl diphosphate synthase. Moreover, we discovered a metabolically available carbon source for terpenoid formation in plants that is accessible via IPK overexpression. This metabolite reactivation approach offers new strategies for metabolic engineering of terpenoid production.
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Pérez, Lucía, Rui Alves, Laura Perez-Fons, Alfonso Albacete, Gemma Farré, Erika Soto, Ester Vilaprinyó, et al. "Multilevel interactions between native and ectopic isoprenoid pathways affect global metabolism in rice." Transgenic Research 31, no. 2 (February 24, 2022): 249–68. http://dx.doi.org/10.1007/s11248-022-00299-6.
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AbstractIsoprenoids are natural products derived from isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). In plants, these precursors are synthesized via the cytosolic mevalonate (MVA) and plastidial methylerythritol phosphate (MEP) pathways. The regulation of these pathways must therefore be understood in detail to develop effective strategies for isoprenoid metabolic engineering. We hypothesized that the strict regulation of the native MVA pathway could be circumvented by expressing an ectopic plastidial MVA pathway that increases the accumulation of IPP and DMAPP in plastids. We therefore introduced genes encoding the plastid-targeted enzymes HMGS, tHMGR, MK, PMK and MVD and the nuclear-targeted transcription factor WR1 into rice and evaluated the impact of their endosperm-specific expression on (1) endogenous metabolism at the transcriptomic and metabolomic levels, (2) the synthesis of phytohormones, carbohydrates and fatty acids, and (3) the macroscopic phenotype including seed morphology. We found that the ectopic plastidial MVA pathway enhanced the expression of endogenous cytosolic MVA pathway genes while suppressing the native plastidial MEP pathway, increasing the production of certain sterols and tocopherols. Plants carrying the ectopic MVA pathway only survived if WR1 was also expressed to replenish the plastid acetyl-CoA pool. The transgenic plants produced higher levels of fatty acids, abscisic acid, gibberellins and lutein, reflecting crosstalk between phytohormones and secondary metabolism.
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Wang, Jin-Zheng, Yongxing Lei, Yanmei Xiao, Xiang He, Jiubo Liang, Jishan Jiang, Shangzhi Dong, et al. "Uncovering the functional residues ofArabidopsisisoprenoid biosynthesis enzyme HDS." Proceedings of the National Academy of Sciences 117, no. 1 (December 26, 2019): 355–61. http://dx.doi.org/10.1073/pnas.1916434117.
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The methylerythritol phosphate (MEP) pathway is responsible for producing isoprenoids, metabolites with essential functions in the bacterial kingdom and plastid-bearing organisms including plants and Apicomplexa. Additionally, the MEP-pathway intermediate methylerythritol cyclodiphosphate (MEcPP) serves as a plastid-to-nucleus retrograde signal. A suppressor screen of the high MEcPP accumulating mutant plant (ceh1) led to the isolation of 3 revertants (designatedRceh1–3) resulting from independent intragenic substitutions of conserved amino acids in the penultimate MEP-pathway enzyme, hydroxymethylbutenyl diphosphate synthase (HDS). The revertants accumulate varying MEcPP levels, lower than that ofceh1, and exhibit partial or full recovery of MEcPP-mediated phenotypes, including stunted growth and induced expression of stress response genes and the corresponding metabolites. Structural modeling of HDS and ligand docking spatially position the substituted residues at the MEcPP binding pocket and cofactor binding domain of the enzyme. Complementation assays confirm the role of these residues in suppressing theceh1mutant phenotypes, albeit to different degrees. In vitro enzyme assays of wild type and HDS variants exhibit differential activities and reveal an unanticipated mismatch between enzyme kinetics and the in vivo MEcPP levels in the correspondingRcehlines. Additional analyses attribute the mismatch, in part, to the abundance of the first and rate-limiting MEP-pathway enzyme, DXS, and further suggest MEcPP as a rheostat for abundance of the upstream enzyme instrumental in fine-tuning of the pathway flux. Collectively, this study identifies critical residues of a key MEP-pathway enzyme, HDS, valuable for synthetic engineering of isoprenoids, and as potential targets for rational design of antiinfective drugs.
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Rohmer, Michel. "Diversity in isoprene unit biosynthesis: The methylerythritol phosphate pathway in bacteria and plastids." Pure and Applied Chemistry 79, no. 4 (January 1, 2007): 739–51. http://dx.doi.org/10.1351/pac200779040739.
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The long-overlooked methylerythritol phosphate (MEP) pathway represents an alternative to the mevalonate route for the formation of isoprene units. It is found in most bacteria as well as in the plastids of all phototrophic organisms. A selection of significant steps of its discovery and elucidation are presented in this contribution, as well as a complete hypothetical biogenetic scheme for the last reduction step.
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Jin, Shi Kun, and Shou Jing Zhao. "Recent Advances in Study of Ginsenoside Biosynthetic Pathway in Panax ginseng." Advanced Materials Research 773 (September 2013): 368–73. http://dx.doi.org/10.4028/www.scientific.net/amr.773.368.
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Ginsenosides, the major bioactive ingredients of P. ginseng can improve the anti-disease abilities of human being, and generate significant social and economic benefits. However, along with gradually or rapidly or dramatically increasing demand of the ginsenosides, extensive studies have focused on regulating the ginsenoside biosynthetic pathway on a genetic level. This review provides the latest research progress on biosynthetic pathway of ginsenosides, including the mevalonate (MVA) and the methylerythritol phosphate (MEP) pathway, which is newly discovered and located in P. ginseng. Moreover, it also indicated lanosterol synthase metabolic flux present in P. ginseng.
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Roth, Jared H., and Valerie C. A. Ward. "Production of Astaxanthin Using CBFD1/HFBD1 from Adonis aestivalis and the Isopentenol Utilization Pathway in Escherichia coli." Bioengineering 10, no. 9 (September 1, 2023): 1033. http://dx.doi.org/10.3390/bioengineering10091033.
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Astaxanthin is a powerful antioxidant and is used extensively as an animal feed additive and nutraceutical product. Here, we report the use of the β-carotene hydroxylase (CBFD1) and the β-carotene ketolase (HBFD1) from Adonis aestivalis, a flowering plant, to produce astaxanthin in E. coli equipped with the P. agglomerans β-carotene pathway and an over-expressed 4-methylerythritol-phosphate (MEP) pathway or the isopentenol utilization pathway (IUP). Introduction of the over-expressed MEP pathway and the IUP resulted in a 3.2-fold higher carotenoid content in LB media at 36 h post-induction compared to the strain containing only the endogenous MEP. However, in M9 minimal media, the IUP pathway dramatically outperformed the over-expressed MEP pathway with an 11-fold increase in total carotenoids produced. The final construct split the large operon into two smaller operons, both with a T7 promoter. This resulted in slightly lower productivity (70.0 ± 8.1 µg/g·h vs. 53.5 ± 3.8 µg/g·h) compared to the original constructs but resulted in the highest proportion of astaxanthin in the extracted carotenoids (73.5 ± 0.2%).
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Nguyen, Anh Duc, Diep Ngoc Pham, Tin Hoang Trung Chau, and Eun Yeol Lee. "Enhancing Sesquiterpenoid Production from Methane via Synergy of the Methylerythritol Phosphate Pathway and a Short-Cut Route to 1-Deoxy-D-xylulose 5-Phosphate in Methanotrophic Bacteria." Microorganisms 9, no. 6 (June 7, 2021): 1236. http://dx.doi.org/10.3390/microorganisms9061236.
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Sesquiterpenoids are one of the most diverse classes of isoprenoids which exhibit numerous potentials in industrial biotechnology. The methanotrophs-based methane bioconversion is a promising approach for sustainable production of chemicals and fuels from methane. With intrinsic high carbon flux though the ribulose monophosphate cycle in Methylotuvimicrobium alcaliphilum 20Z, we demonstrated here that employing a short-cut route from ribulose 5-phosphate to 1-deoxy-d-xylulose 5-phosphate (DXP) could enable a more efficient isoprenoid production via the methylerythritol 4-phosphate (MEP) pathway, using α-humulene as a model compound. An additional 2.8-fold increase in α-humulene production yield was achieved by the fusion of the nDXP enzyme and DXP reductase. Additionally, we utilized these engineering strategies for the production of another sesquiterpenoid, α-bisabolene. The synergy of the nDXP and MEP pathways improved the α-bisabolene titer up to 12.24 ± 0.43 mg/gDCW, twofold greater than that of the initial strain. This study expanded the suite of sesquiterpenoids that can be produced from methane and demonstrated the synergistic uses of the nDXP and MEP pathways for improving sesquiterpenoid production in methanotrophic bacteria.
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Gastaldo, Lipko, Motsch, Adam, Schaeffer, and Rohmer. "Biosynthesis of Isoprene Units in Euphorbia lathyris Laticifers vs. Other Tissues: MVA and MEP Pathways, Compartmentation and Putative Endophytic Fungi Contribution." Molecules 24, no. 23 (November 26, 2019): 4322. http://dx.doi.org/10.3390/molecules24234322.
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Euphorbia species are characterized by a net of laticifers producing large amounts of triterpenes. These hydrocarbon-like metabolites can be converted into fuel by the methods of the oil industry. Euphorbia lathyris is easily grown at an industrial scale. In an attempt to increase its triterpene production, the metabolic pathways leading to isoprenoid were investigated by incorporation of 13C labeled glucose and mevalonate and 2H labeled deoxyxylulose as well as by natural abundance isotope ratio GC-MS. Latex triterpenes are exclusively synthesized via the mevalonate (MVA) pathway: this may orient future search for improving the triterpene production in E. lathyris. Phytosterols and their precursors are mainly derived from MVA pathway with a slight contribution of the methylerythritol phosphate (MEP) pathway, whereas phytol is issued from MEP pathway with a minor contribution of the MVA pathway: this is in accordance with the metabolic cross-talk between cytosolic and plastidial compartments in plants. In addition, hopenol B behaved differently from the other latex triterpenes. Its 13C isotope abundance after incorporation of 13C labeled glucose and its natural abundance δ2H signature clearly differed from those of the other latex triterpenes indicating another metabolic origin and suggesting that it may be synthesized by an endophytic fungus.
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Mueller, C., J. Schwender, J. Zeidler, and H. K. Lichtenthaler. "Properties and inhibition of the first two enzymes of the non-mevalonate pathway of isoprenoid biosynthesis." Biochemical Society Transactions 28, no. 6 (December 1, 2000): 792–93. http://dx.doi.org/10.1042/bst0280792.
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Enzymes of the 1-deoxy-D-xylulose 5-phosphate/2-C-methylerythritol 4-phosphate (DOXP/MEP) pathway are targets for new herbicides and antibacterial drugs. Until now, no inhibitors for the DOXP synthase have been known of. We show that one of the breakdown products of the herbicide clomazone affects the DOXP synthase. One inhibitor of the non-mevalonate pathway, fosmidomycin, blocks the DOXP reductoisomerase (DXR) of plants and bacteria. The I50 values of plants are, however, higher than those found for the DXR of Escherichia coli. The DXR of plants, isolated from barley seedlings, shows a pH optimum of 8.1, which is typical for enzymes active in the chloroplast stroma.
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Rohmer, M. "The mevalonate-independent methylerythritol 4-phosphate (MEP) pathway for isoprenoid biosynthesis, including carotenoids." Pure and Applied Chemistry 71, no. 12 (January 1, 1999): 2279–84. http://dx.doi.org/10.1351/pac199971122279.
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Pierce, Phillip G., Brian E. Hartnett, Tosha M. Laughlin, Joy M. Blain, Stephen J. Mayclin, Madison J. Bolejack, Janette B. Myers, et al. "Crystal structure and biophysical characterization of IspD from Burkholderia thailandensis and Mycobacterium paratuberculosis." Acta Crystallographica Section F Structural Biology Communications 80, no. 2 (January 31, 2024): 43–51. http://dx.doi.org/10.1107/s2053230x24000621.
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The methylerythritol phosphate (MEP) pathway is a metabolic pathway that produces the isoprenoids isopentyl pyrophosphate and dimethylallyl pyrophosphate. Notably, the MEP pathway is present in bacteria and not in mammals, which makes the enzymes of the MEP pathway attractive targets for discovering new anti-infective agents due to the reduced chances of off-target interactions leading to side effects. There are seven enzymes in the MEP pathway, the third of which is IspD. Two crystal structures of Burkholderia thailandensis IspD (BtIspD) were determined: an apo structure and that of a complex with cytidine triphosphate (CTP). Comparison of the CTP-bound BtIspD structure with the apo structure revealed that CTP binding stabilizes the loop composed of residues 13–19. The apo structure of Mycobacterium paratuberculosis IspD (MpIspD) is also reported. The melting temperatures of MpIspD and BtIspD were evaluated by circular dichroism. The moderate T m values suggest that a thermal shift assay may be feasible for future inhibitor screening. Finally, the binding affinity of CTP for BtIspD was evaluated by isothermal titration calorimetry. These structural and biophysical data will aid in the discovery of IspD inhibitors.
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Niu, Zhipeng, Shu Ye, Jiaojiao Liu, Mengyu Lyu, Lilan Xue, Muxiao Li, Congcong Lyu, Junlong Zhao, and Bang Shen. "Two apicoplast dwelling glycolytic enzymes provide key substrates for metabolic pathways in the apicoplast and are critical for Toxoplasma growth." PLOS Pathogens 18, no. 11 (November 30, 2022): e1011009. http://dx.doi.org/10.1371/journal.ppat.1011009.
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Many apicomplexan parasites harbor a non-photosynthetic plastid called the apicoplast, which hosts important metabolic pathways like the methylerythritol 4-phosphate (MEP) pathway that synthesizes isoprenoid precursors. Yet many details in apicoplast metabolism are not well understood. In this study, we examined the physiological roles of four glycolytic enzymes in the apicoplast of Toxoplasma gondii. Many glycolytic enzymes in T. gondii have two or more isoforms. Endogenous tagging each of these enzymes found that four of them were localized to the apicoplast, including pyruvate kinase2 (PYK2), phosphoglycerate kinase 2 (PGK2), triosephosphate isomerase 2 (TPI2) and phosphoglyceraldehyde dehydrogenase 2 (GAPDH2). The ATP generating enzymes PYK2 and PGK2 were thought to be the main energy source of the apicoplast. Surprisingly, deleting PYK2 and PGK2 individually or simultaneously did not cause major defects on parasite growth or virulence. In contrast, TPI2 and GAPDH2 are critical for tachyzoite proliferation. Conditional depletion of TPI2 caused significant reduction in the levels of MEP pathway intermediates and led to parasite growth arrest. Reconstitution of another isoprenoid precursor synthesis pathway called the mevalonate pathway in the TPI2 depletion mutant partially rescued its growth defects. Similarly, knocking down the GAPDH2 enzyme that produces NADPH also reduced isoprenoid precursor synthesis through the MEP pathway and inhibited parasite proliferation. In addition, it reduced de novo fatty acid synthesis in the apicoplast. Together, these data suggest a model that the apicoplast dwelling TPI2 provides carbon source for the synthesis of isoprenoid precursor, whereas GAPDH2 supplies reducing power for pathways like MEP, fatty acid synthesis and ferredoxin redox system in T. gondii. As such, both enzymes are critical for parasite growth and serve as potential targets for anti-toxoplasmic intervention designs. On the other hand, the dispensability of PYK2 and PGK2 suggest additional sources for energy in the apicoplast, which deserves further investigation.
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Knak, Talea, Mona A. Abdullaziz, Stefan Höfmann, Leandro A. Alves Avelar, Saskia Klein, Matthew Martin, Markus Fischer, Nobutada Tanaka, and Thomas Kurz. "Over 40 Years of Fosmidomycin Drug Research: A Comprehensive Review and Future Opportunities." Pharmaceuticals 15, no. 12 (December 14, 2022): 1553. http://dx.doi.org/10.3390/ph15121553.
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To address the continued rise of multi-drug-resistant microorganisms, the development of novel drugs with new modes of action is urgently required. While humans biosynthesize the essential isoprenoid precursors isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) via the established mevalonate pathway, pathogenic protozoa and certain pathogenic eubacteria use the less well-known methylerythritol phosphate pathway for this purpose. Important pathogens using the MEP pathway are, for example, Plasmodium falciparum, Mycobacterium tuberculosis, Pseudomonas aeruginosa and Escherichia coli. The enzymes of that pathway are targets for antiinfective drugs that are exempt from target-related toxicity. 2C-Methyl-D-erythritol 4-phosphate (MEP), the second enzyme of the non-mevalonate pathway, has been established as the molecular target of fosmidomycin, an antibiotic that has so far failed to be approved as an anti-infective drug. This review describes the development and anti-infective properties of a wide range of fosmidomycin derivatives synthesized over the last four decades. Here we discuss the DXR inhibitor pharmacophore, which comprises a metal-binding group, a phosphate or phosphonate moiety and a connecting linker. Furthermore, non-fosmidomycin-based DXRi, bisubstrate inhibitors and several prodrug concepts are described. A comprehensive structure–activity relationship (SAR) of nearly all inhibitor types is presented and some novel opportunities for further drug development of DXR inhibitors are discussed.
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Van Nguyen, Truong, So-Wun Kim, Cheol-Woo Min, Ravi Gupta, Gi-Hyun Lee, Jeong-Woo Jang, Divya Rathi, et al. "Optimization of Protein Isolation and Label-Free Quantitative Proteomic Analysis in Four Different Tissues of Korean Ginseng." Plants 10, no. 7 (July 9, 2021): 1409. http://dx.doi.org/10.3390/plants10071409.
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Korean ginseng is one of the most valuable medicinal plants worldwide. However, our understanding of ginseng proteomics is largely limited due to difficulties in the extraction and resolution of ginseng proteins because of the presence of natural contaminants such as polysaccharides, phenols, and glycosides. Here, we compared four different protein extraction methods, namely, TCA/acetone, TCA/acetone–MeOH/chloroform, phenol–TCA/acetone, and phenol–MeOH/chloroform methods. The TCA/acetone–MeOH/chloroform method displayed the highest extraction efficiency, and thus it was used for the comparative proteome profiling of leaf, root, shoot, and fruit by a label-free quantitative proteomics approach. This approach led to the identification of 2604 significantly modulated proteins among four tissues. We could pinpoint differential pathways and proteins associated with ginsenoside biosynthesis, including the methylerythritol 4–phosphate (MEP) pathway, the mevalonate (MVA) pathway, UDP-glycosyltransferases (UGTs), and oxidoreductases (CYP450s). The current study reports an efficient and reproducible method for the isolation of proteins from a wide range of ginseng tissues and provides a detailed organ-based proteome map and a more comprehensive view of enzymatic alterations in ginsenoside biosynthesis.
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Eoh, Hyungjin, Amanda C. Brown, Lori Buetow, William N. Hunter, Tanya Parish, Devinder Kaur, Patrick J. Brennan, and Dean C. Crick. "Characterization of the Mycobacterium tuberculosis 4-Diphosphocytidyl-2-C-Methyl-d-Erythritol Synthase: Potential for Drug Development." Journal of Bacteriology 189, no. 24 (October 5, 2007): 8922–27. http://dx.doi.org/10.1128/jb.00925-07.
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ABSTRACT Mycobacterium tuberculosis utilizes the methylerythritol phosphate (MEP) pathway for biosynthesis of isopentenyl diphosphate and its isomer, dimethylallyl diphosphate, precursors of all isoprenoid compounds. This pathway is of interest as a source of new drug targets, as it is absent from humans and disruption of the responsible genes has shown a lethal phenotype for Escherichia coli. In the MEP pathway, 4-diphosphocytidyl-2-C-methyl-d-erythritol is formed from 2-C-methyl-d-erythritol 4-phosphate (MEP) and CTP in a reaction catalyzed by a 4-diphosphocytidyl-2-C-methyl-d-erythritol synthase (IspD). In the present work, we demonstrate that Rv3582c is essential for M. tuberculosis: Rv3582c has been cloned and expressed, and the encoded protein has been purified. The purified M. tuberculosis IspD protein was capable of catalyzing the formation of 4-diphosphocytidyl-2-C-methyl-d-erythritol in the presence of MEP and CTP. The enzyme was active over a broad pH range (pH 6.0 to 9.0), with peak activity at pH 8.0. The activity was absolutely dependent upon divalent cations, with 20 mM Mg2+ being optimal, and replacement of CTP with other nucleotide 5′-triphosphates did not support activity. Under the conditions tested, M. tuberculosis IspD had Km values of 58.5 μM for MEP and 53.2 μM for CTP. Calculated k cat and k cat/Km values were 0.72 min−1 and 12.3 mM−1 min−1 for MEP and 1.0 min−1 and 18.8 mM−1 min−1 for CTP, respectively.
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Howe, Ruth, Megan Kelly, John Jimah, Dana Hodge, and Audrey R. Odom. "Isoprenoid Biosynthesis Inhibition Disrupts Rab5 Localization and Food Vacuolar Integrity in Plasmodium falciparum." Eukaryotic Cell 12, no. 2 (December 7, 2012): 215–23. http://dx.doi.org/10.1128/ec.00073-12.
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ABSTRACT The antimalarial agent fosmidomycin is a validated inhibitor of the nonmevalonate isoprenoid biosynthesis (methylerythritol 4-phosphate [MEP]) pathway in the malaria parasite, Plasmodium falciparum . Since multiple classes of prenyltransferase inhibitors kill P. falciparum , we hypothesized that protein prenylation was one of the essential functions of this pathway. We found that MEP pathway inhibition with fosmidomycin reduces protein prenylation, confirming that de novo isoprenoid biosynthesis produces the isoprenyl substrates for protein prenylation. One important group of prenylated proteins is small GTPases, such as Rab family members, which mediate cellular vesicular trafficking. We have found that Rab5 proteins dramatically mislocalize upon fosmidomycin treatment, consistent with a loss of protein prenylation. Fosmidomycin treatment caused marked defects in food vacuolar morphology and integrity, consistent with a defect in Rab-mediated vesicular trafficking. These results provide insights to the biological functions of isoprenoids in malaria parasites and may assist the rational selection of secondary agents that will be useful in combination therapy with new isoprenoid biosynthesis inhibitors.
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Armstrong, Christopher M., David J. Meyers, Leah S. Imlay, Caren Freel Meyers, and Audrey R. Odom. "Resistance to the Antimicrobial Agent Fosmidomycin and an FR900098 Prodrug through Mutations in the Deoxyxylulose Phosphate Reductoisomerase Gene (dxr)." Antimicrobial Agents and Chemotherapy 59, no. 9 (June 29, 2015): 5511–19. http://dx.doi.org/10.1128/aac.00602-15.
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ABSTRACTThere is a pressing need for new antimicrobial therapies to combat globally important drug-resistant human pathogens, includingPlasmodium falciparummalarial parasites,Mycobacterium tuberculosis, and Gram-negative bacteria, includingEscherichia coli. These organisms all possess the essential methylerythritol phosphate (MEP) pathway of isoprenoid biosynthesis, which is not found in humans. The first dedicated enzyme of the MEP pathway, 1-deoxy-d-xylulose 5-phosphate reductoisomerase (Dxr), is inhibited by the phosphonic acid antibiotic fosmidomycin and its analogs, including theN-acetyl analog FR900098 and the phosphoryl analog fosfoxacin. In order to identify mutations indxrthat confer resistance to these drugs, a library ofE. colidxrmutants was screened at lethal fosmidomycin doses. The most resistant allele (with the S222T mutation) alters the fosmidomycin-binding site of Dxr. The expression of this resistant allele increases bacterial resistance to fosmidomycin and other fosmidomycin analogs by 10-fold. These observations confirm that the primary cellular target of fosmidomycin is Dxr. Furthermore, cell lines expressing Dxr-S222T will be a powerful tool to confirm the mechanisms of action of future fosmidomycin analogs.
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HOEFFLER, Jean-François, Andréa HEMMERLIN, Catherine GROSDEMANGE-BILLIARD, Thomas J. BACH, and Michel ROHMER. "Isoprenoid biosynthesis in higher plants and in Escherichia coli: on the branching in the methylerythritol phosphate pathway and the independent biosynthesis of isopentenyl diphosphate and dimethylallyl diphosphate." Biochemical Journal 366, no. 2 (September 1, 2002): 573–83. http://dx.doi.org/10.1042/bj20020337.
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In the bacterium Escherichia coli, the mevalonic-acid (MVA)-independent 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway is characterized by two branches leading separately to isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). The signature of this branching is the retention of deuterium in DMAPP and the deuterium loss in IPP after incorporation of 1-[4-2H]deoxy-d-xylulose ([4-2H]DX). Feeding tobacco BY-2 cell-suspension cultures with [4-2H]DX resulted in deuterium retention in the isoprene units derived from DMAPP, as well as from IPP in the plastidial isoprenoids, phytoene and plastoquinone, synthesized via the MEP pathway. This labelling pattern represents direct evidence for the presence of the DMAPP branch of the MEP pathway in a higher plant, and shows that IPP can be synthesized from DMAPP in plant plastids, most probably via a plastidial IPP isomerase.
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Zhu, Jianhua, Pu Wang, Minghua Qian, Chuxin Liang, Jiachen Zi, and Rongmin Yu. "Effect of Levopimaradiene on Terpene Trilactones Biosynthesis and Gene Expression Profiling in Ginkgo biloba Cells." Natural Product Communications 12, no. 7 (July 2017): 1934578X1701200. http://dx.doi.org/10.1177/1934578x1701200701.
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Ginkgolides (GKs) and Bilobalide (BB) are rare terpene trilactones obtained from Ginkgo biloba, but their biosynthetic pathway is still unclear. In this paper, effects of levopimaradiene (LP) on increasing the production of terpene trilactones of G. biloba dedifferentiated cells (DDCs) and cambial meristematic cells (CMCs) were reported. The productions of ginkgolide A (GA) and ginkgolide B (GB) were 1.61 and 1.32 folds larger than that of the control groups when G. biloba DDCs was treated with LP, and the productions of ginkgolide C (GC) and BB reached 234 and 161 μg L−1 after treated with LP for 60 h. The production of GA, GB, GC and BB was 2.03, 1.43, 1.22 and 1.19 folds larger than that of the control groups in LP-treated CMCs groups. The results demonstrated that BB could be produced from the methylerythritol 4-phosphate (MEP) pathway. qRT-PCR experiments showed that LP was a significant precursor manipulated the biosynthesis of terpene trilactones via affecting the transcript levels of several related genes in the MEP pathway.
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Hartmann, Michael, Andrea Hemmerlin, Elisabet Gas-Pascual, Esther Gerber, Denis Tritsch, Michel Rohmer, and Thomas J. Bach. "The effect of MEP pathway and other inhibitors on the intracellular localization of a plasma membrane-targeted, isoprenylable GFP reporter protein in tobacco BY-2 cells." F1000Research 2 (August 12, 2013): 170. http://dx.doi.org/10.12688/f1000research.2-170.v1.
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We have established an in vivo visualization system for the geranylgeranylation of proteins in a stably transformed tobacco BY-2 cell line, based on the expression of a dexamethasone-inducible GFP fused to the carboxy-terminal basic domain of the rice calmodulin CaM61, which naturally bears a CaaL geranylgeranylation motif (GFP-BD-CVIL). By using pathway-specific inhibitors it was demonstrated that inhibition of the methylerythritol phosphate (MEP) pathway with known inhibitors like oxoclomazone and fosmidomycin, as well as inhibition of the protein geranylgeranyltransferase type 1 (PGGT-1), shifted the localization of the GFP-BD-CVIL protein from the membrane to the nucleus. In contrast, the inhibition of the mevalonate (MVA) pathway with mevinolin did not affect the localization. During the present work, this test system has been used to examine the effect of newly designed inhibitors of the MEP pathway and inhibitors of sterol biosynthesis such as squalestatin, terbinafine and Ro48-8071. In addition, we also studied the impact of different post-prenylation inhibitors or those suspected to affect the transport of proteins to the plasma membrane on the localization of the geranylgeranylable fusion protein GFP-BD-CVIL.
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Hartmann, Michael, Andrea Hemmerlin, Elisabet Gas-Pascual, Esther Gerber, Denis Tritsch, Michel Rohmer, and Thomas J. Bach. "The effect of MEP pathway and other inhibitors on the intracellular localization of a plasma membrane-targeted, isoprenylable GFP reporter protein in tobacco BY-2 cells." F1000Research 2 (November 15, 2013): 170. http://dx.doi.org/10.12688/f1000research.2-170.v2.
Full textAbstract:
We have established an in vivo visualization system for the geranylgeranylation of proteins in a stably transformed tobacco BY-2 cell line, based on the expression of a dexamethasone-inducible GFP fused to the carboxy-terminal basic domain of the rice calmodulin CaM61, which naturally bears a CaaL geranylgeranylation motif (GFP-BD-CVIL). By using pathway-specific inhibitors it was demonstrated that inhibition of the methylerythritol phosphate (MEP) pathway with known inhibitors like oxoclomazone and fosmidomycin, as well as inhibition of the protein geranylgeranyltransferase type 1 (PGGT-1), shifted the localization of the GFP-BD-CVIL protein from the membrane to the nucleus. In contrast, the inhibition of the mevalonate (MVA) pathway with mevinolin did not affect the localization. During the present work, this test system has been used to examine the effect of newly designed inhibitors of the MEP pathway and inhibitors of sterol biosynthesis such as squalestatin, terbinafine and Ro48-8071. In addition, we also studied the impact of different post-prenylation inhibitors or those suspected to affect the transport of proteins to the plasma membrane on the localization of the geranylgeranylable fusion protein GFP-BD-CVIL.
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Liu, Yu, Hui Zhang, Shivshankar Umashankar, Xu Liang, Hui Lee, Sanjay Swarup, and Choon Ong. "Characterization of Plant Volatiles Reveals Distinct Metabolic Profiles and Pathways among 12 Brassicaceae Vegetables." Metabolites 8, no. 4 (December 14, 2018): 94. http://dx.doi.org/10.3390/metabo8040094.
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Plants emit characteristic organic volatile compounds (VOCs) with diverse biological/ecological functions. However, the links between plant species/varieties and their phytochemical emission profiles remain elusive. Here, we developed a direct headspace solid-phase microextraction (HS-SPME) technique and combined with non-targeted gas chromatography‒high-resolution mass spectrometry (GC-HRMS) platform to investigate the VOCs profiles of 12 common Brassicaceae vegetables (watercress, rocket, Brussels sprouts, broccoli, kai lan, choy sum, pak choi, cabbage, Chinese cabbage, cauliflower, radish and cherry radish). The direct HS-SPME sampling approach enabled reproducible capture of the rapid-emitting VOCs upon plant tissue disruption. The results revealed extensive variation in VOCs profiles among the 12 Brassicaceae vegetables. Furthermore, principal component analysis (PCA) showed that the VOC profiles could clearly distinguish the 12 Brassicaceae vegetables, and that these profiles well reflected the classical morphological classification. After multivariate statistical analysis, 44 VOCs with significant differences among the Brassicaceae vegetables were identified. Pathway analysis showed that three secondary metabolism pathways, including the fatty acid pathway, methylerythritol phosphate (MEP) pathway and glucosinolate (GLS) pathway, behave distinctively in these vegetables. These three pathways are responsible for the generation and emission of green leaf volatiles (GLVs), terpenes and isothiocyanates (ITCs), respectively. Correlation analysis further showed that volatile metabolites formed via the common pathway had significantly positive correlations, whereas metabolites from different pathways had either non-significant or significantly negative correlations. Genetic influences on these metabolites across various vegetable types were also evaluated. These findings extend our phytochemical knowledge of the 12 edible Brassicaceae vegetables and provide useful information on their secondary metabolism.
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Zhu, Peihuang, Yu Chen, Fan Wu, Miaojing Meng, and Kongshu Ji. "Expression and promoter analysis of MEP pathway enzyme-encoding genes in Pinus massoniana Lamb." PeerJ 10 (April 12, 2022): e13266. http://dx.doi.org/10.7717/peerj.13266.
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The methylerythritol phosphate (MEP) pathway provides the universal basic blocks for the biosynthesis of terpenoids and plays a critical role in the growth and development of higher plants. Pinus massoniana is the most valuable oleoresin producer tree with an extensive terrestrial range. It has the potential to produce more oleoresin with commercial value, while being resistant to pine wood nematode (PWN) disease. For this study, eleven MEP pathway associated enzyme-encoding genes and ten promoters were isolated from P. massoniana. Three PmDXS and two PmHDR existed as multi-copy genes, whereas the other six genes existed as single copies. All eleven of these MEP enzymes exhibited chloroplast localization with transient expression. Most of the MEP genes showed higher expression in the needles, while PmDXS2, PmDXS3, and PmHDR1 had high expression in the roots. The expressions of a few MEP genes could be induced under exogenous elicitor conditions. The functional complementation in a dxs-mutant Escherichia coli strain showed the DXS enzymatic activities of the three PmDXSs. High throughput TAIL PCR was employed to obtain the upstream sequences of the genes encoding for enzymes in the MEP pathway, whereby abundant light responsive cis-elements and transcription factor (TF) binding sites were identified within the ten promoters. This study provides a theoretical basis for research on the functionality and transcriptional regulation of MEP enzymes, as well as a potential strategy for high-resin generation and improved genetic resistance in P. massoniana.
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Jezewski, Andrew J., Ann M. Guggisberg, Dana M. Hodge, Naomi Ghebremichael, Gavin Nicholas John, Lisa K. McLellan, and Audrey Ragan Odom John. "GAPDH mediates drug resistance and metabolism in Plasmodium falciparum malaria parasites." PLOS Pathogens 18, no. 9 (September 14, 2022): e1010803. http://dx.doi.org/10.1371/journal.ppat.1010803.
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Efforts to control the global malaria health crisis are undermined by antimalarial resistance. Identifying mechanisms of resistance will uncover the underlying biology of the Plasmodium falciparum malaria parasites that allow evasion of our most promising therapeutics and may reveal new drug targets. We utilized fosmidomycin (FSM) as a chemical inhibitor of plastidial isoprenoid biosynthesis through the methylerythritol phosphate (MEP) pathway. We have thus identified an unusual metabolic regulation scheme in the malaria parasite through the essential glycolytic enzyme, glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Two parallel genetic screens converged on independent but functionally analogous resistance alleles in GAPDH. Metabolic profiling of FSM-resistant gapdh mutant parasites indicates that neither of these mutations disrupt overall glycolytic output. While FSM-resistant GAPDH variant proteins are catalytically active, they have reduced assembly into the homotetrameric state favored by wild-type GAPDH. Disrupted oligomerization of FSM-resistant GAPDH variant proteins is accompanied by altered enzymatic cooperativity and reduced susceptibility to inhibition by free heme. Together, our data identifies a new genetic biomarker of FSM-resistance and reveals the central role of GAPDH in MEP pathway control and antimalarial sensitivity.
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Lu, Zhifang, Biying Wang, Zhiyu Qiu, Ruiling Zhang, Jimin Zheng, and Zongchao Jia. "YdfD, a Lysis Protein of the Qin Prophage, Is a Specific Inhibitor of the IspG-Catalyzed Step in the MEP Pathway of Escherichia coli." International Journal of Molecular Sciences 23, no. 3 (January 29, 2022): 1560. http://dx.doi.org/10.3390/ijms23031560.
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Bacterial cryptic prophage (defective prophage) genes are known to drastically influence host physiology, such as causing cell growth arrest or lysis, upon expression. Many phages encode lytic proteins to destroy the cell envelope. As natural antibiotics, only a few lysis target proteins were identified. ydfD is a lytic gene from the Qin cryptic prophage that encodes a 63-amino-acid protein, the ectopic expression of which in Escherichia coli can cause nearly complete cell lysis rapidly. The bacterial 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway is responsible for synthesizing the isoprenoids uniquely required for sustaining bacterial growth. In this study, we provide evidence that YdfD can interact with IspG, a key enzyme involved in the MEP pathway, both in vivo and in vitro. We show that intact YdfD is required for the interaction with IspG to perform its lysis function and that the mRNA levels of ydfD increase significantly under certain stress conditions. Crucially, the cell lysis induced by YdfD can be abolished by the overexpression of ispG or the complementation of the IspG enzyme catalysis product methylerythritol 2,4-cyclodiphosphate. We propose that YdfD from the Qin cryptic prophage inhibits IspG to block the MEP pathway, leading to a compromised cell membrane and cell wall biosynthesis and eventual cell lysis.
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Dong, Miaoyin, Jinjuan Li, Delong Yang, Mengfei Li, and Jianhe Wei. "Biosynthesis and Pharmacological Activities of Flavonoids, Triterpene Saponins and Polysaccharides Derived from Astragalus membranaceus." Molecules 28, no. 13 (June 27, 2023): 5018. http://dx.doi.org/10.3390/molecules28135018.
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Astragalus membranaceus (A. membranaceus), a well-known traditional herbal medicine, has been widely used in ailments for more than 2000 years. The main bioactive compounds including flavonoids, triterpene saponins and polysaccharides obtained from A. membranaceus have shown a wide range of biological activities and pharmacological effects. These bioactive compounds have a significant role in protecting the liver, immunomodulation, anticancer, antidiabetic, antiviral, antiinflammatory, antioxidant and anti-cardiovascular activities. The flavonoids are initially synthesized through the phenylpropanoid pathway, followed by catalysis with corresponding enzymes, while the triterpenoid saponins, especially astragalosides, are synthesized through the universal upstream pathways of mevalonate (MVA) and methylerythritol phosphate (MEP), and the downstream pathway of triterpenoid skeleton formation and modification. Moreover, the Astragalus polysaccharide (APS) possesses multiple pharmacological activities. In this review, we comprehensively discussed the biosynthesis pathway of flavonoids and triterpenoid saponins, and the structural features of polysaccharides in A. membranaceus. We further systematically summarized the pharmacological effects of bioactive ingredients in A. membranaceus, which laid the foundation for the development of clinical candidate agents. Finally, we proposed potential strategies of heterologous biosynthesis to improve the industrialized production and sustainable supply of natural products with pharmacological activities from A. membranaceus, thereby providing an important guide for their future development trend.
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Dini, Irene, Roberta Marra, Pierpaolo Cavallo, Angela Pironti, Immacolata Sepe, Jacopo Troisi, Giovanni Scala, Pasquale Lombari, and Francesco Vinale. "Trichoderma Strains and Metabolites Selectively Increase the Production of Volatile Organic Compounds (VOCs) in Olive Trees." Metabolites 11, no. 4 (March 31, 2021): 213. http://dx.doi.org/10.3390/metabo11040213.
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Plants emit volatile organic compounds (VOCs) that induce metabolomic, transcriptomic, and behavioral reactions in receiver organisms, including insect pollinators and herbivores. VOCs’ composition and concentration may influence plant-insect or plant-plant interactions and affect soil microbes that may interfere in plant-plant communication. Many Trichoderma fungi act as biocontrol agents of phytopathogens and plant growth promoters. Moreover, they can stimulate plant defense mechanisms against insect pests. This study evaluated VOCs’ emission by olive trees (Olea europaea L.) when selected Trichoderma fungi or metabolites were used as soil treatments. Trichoderma harzianum strains M10, T22, and TH1, T. asperellum strain KV906, T. virens strain GV41, and their secondary metabolites harzianic acid (HA), and 6-pentyl-α-pyrone (6PP) were applied to olive trees. Charcoal cartridges were employed to adsorb olive VOCs, and gas chromatography mass spectrometry (GC-MS) analysis allowed their identification and quantification. A total of 45 volatile compounds were detected, and among these, twenty-five represented environmental pollutants and nineteen compounds were related to olive plant emission. Trichoderma strains and metabolites differentially enhanced VOCs production, affecting three biosynthetic pathways: methylerythritol 1-phosphate (MEP), lipid-signaling, and shikimate pathways. Multivariate analysis models showed a characteristic fingerprint of each plant-fungus/metabolite relationship, reflecting a different emission of VOCs by the treated plants. Specifically, strain M10 and the metabolites 6PP and HA enhanced the monoterpene syntheses by controlling the MEP pathway. Strains GV41, KV906, and the metabolite HA stimulated the hydrocarbon aldehyde formation (nonanal) by regulating the lipid-signaling pathway. Finally, Trichoderma strains GV41, M10, T22, TH1, and the metabolites HA and 6PP improve aromatic syntheses at different steps of the shikimate pathway.
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Crispim, Marcell, Ignasi Bofill Verdaguer, Agustín Hernández, Thales Kronenberger, Àngel Fenollar, Lydia Fumiko Yamaguchi, María Pía Alberione, et al. "Beyond the MEP Pathway: A novel kinase required for prenol utilization by malaria parasites." PLOS Pathogens 20, no. 1 (January 26, 2024): e1011557. http://dx.doi.org/10.1371/journal.ppat.1011557.
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A proposed treatment for malaria is a combination of fosmidomycin and clindamycin. Both compounds inhibit the methylerythritol 4-phosphate (MEP) pathway, the parasitic source of farnesyl and geranylgeranyl pyrophosphate (FPP and GGPP, respectively). Both FPP and GGPP are crucial for the biosynthesis of several essential metabolites such as ubiquinone and dolichol, as well as for protein prenylation. Dietary prenols, such as farnesol (FOH) and geranylgeraniol (GGOH), can rescue parasites from MEP inhibitors, suggesting the existence of a missing pathway for prenol salvage via phosphorylation. In this study, we identified a gene in the genome of P. falciparum, encoding a transmembrane prenol kinase (PolK) involved in the salvage of FOH and GGOH. The enzyme was expressed in Saccharomyces cerevisiae, and its FOH/GGOH kinase activities were experimentally validated. Furthermore, conditional knockout parasites (Δ-PolK) were created to investigate the biological importance of the FOH/GGOH salvage pathway. Δ-PolK parasites were viable but displayed increased susceptibility to fosmidomycin. Their sensitivity to MEP inhibitors could not be rescued by adding prenols. Additionally, Δ-PolK parasites lost their capability to utilize prenols for protein prenylation. Experiments using culture medium supplemented with whole/delipidated human plasma in transgenic parasites revealed that human plasma has components that can diminish the effectiveness of fosmidomycin. Mass spectrometry tests indicated that both bovine supplements used in culture and human plasma contain GGOH. These findings suggest that the FOH/GGOH salvage pathway might offer an alternate source of isoprenoids for malaria parasites when de novo biosynthesis is inhibited. This study also identifies a novel kind of enzyme related to isoprenoid metabolism.
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Gawriljuk, Victor Oliveira, Rick Oerlemans, Robin M. Gierse, Riya Jotwani, Anna K. H. Hirsch, and Matthew R. Groves. "Structure of Mycobacterium tuberculosis 1-Deoxy-D-Xylulose 5-Phosphate Synthase in Complex with Butylacetylphosphonate." Crystals 13, no. 5 (April 27, 2023): 737. http://dx.doi.org/10.3390/cryst13050737.
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Stagnation in the development of new antibiotics emphasizes the need for the discovery of drugs with novel modes of action that can tackle antibiotic resistance. Contrary to humans, most bacteria use the methylerythritol phosphate (MEP) pathway to synthesize crucial isoprenoid precursors. 1-deoxy-D-xylulose 5-phosphate synthase (DXPS) catalyzes the first and rate-limiting step of the pathway, making it an attractive target. Alkylacetylphosphonates (alkylAPs) are a class of pyruvate mimicking DXPS inhibitors that react with thiamin diphosphate (ThDP) to form a stable phosphonolactyl (PLThDP) adduct. Here, we present the first M. tuberculosis DXPS crystal structure in complex with an inhibitor (butylacetylphosphonate (BAP)) using a construct with improved crystallization properties. The 1.6 Å structure shows that the BAP adduct interacts with catalytically important His40 and several other conserved residues of the active site. In addition, a glycerol molecule, present in the D-glyceraldehyde 3-phosphate (D-GAP) binding site and within 4 Å of the BAP adduct, indicates that there is space to extend and develop more potent alkylAPs. The structure reveals the BAP binding mode and provides insights for enhancing the activity of alkylAPs against M. tuberculosis, aiding in the development of novel antibiotics.
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Li, Yuchan, Jun Zhao, Hua Chen, Yanping Mao, Yuping Yang, Liang Feng, Chuanxin Mo, Lin Huang, Dabin Hou, and Ma Yu. "Transcriptome Level Reveals the Triterpenoid Saponin Biosynthesis Pathway of Bupleurum falcatum L." Genes 13, no. 12 (November 29, 2022): 2237. http://dx.doi.org/10.3390/genes13122237.
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Bupleurum falcatum L. is frequently used in traditional herbal medicine in Asia. Saikosaponins (SSs) are the main bioactive ingredients of B. falcatum, but the biosynthetic pathway of SSs is unclear, and the biosynthesis of species-specific phytometabolites is little known. Here we resolved the transcriptome profiles of B. falcatum to identify candidate genes that might be involved in the biosynthesis of SSs. By isoform sequencing (Iso-Seq) analyses of the whole plant, a total of 26.98 Gb of nucleotides were obtained and 124,188 unigenes were identified, and 81,594 unigenes were successfully annotated. A total of 1033 unigenes of 20 families related to the mevalonate (MVA) pathway and methylerythritol phosphate (MEP) pathway of the SS biosynthetic pathway were identified. The WGCNA (weighted gene co-expression network analysis) of these unigenes revealed that only the co-expression module of MEmagenta, which contained 343 unigenes, was highly correlated with the biosynthesis of SSs. Comparing differentially expressed gene analysis and the WGCNA indicated that 130 out of 343 genes of the MEmagenta module exhibited differential expression levels, and genes with the most “hubness” within this module were predicted. Manipulation of these genes might improve the biosynthesis of SSs.
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Sripinyowanich, Siriporn, Sahanat Petchsri, Pumipat Tongyoo, Taek-Kyun Lee, Sukchan Lee, and Won Kyong Cho. "Comparative Transcriptomic Analysis of Genes in the 20-hydroxyecdysone Biosynthesis in the Fern Microsorum scolopendria Towards Challenges with Foliar Application of Chitosan." International Journal of Molecular Sciences 24, no. 3 (January 25, 2023): 2397. http://dx.doi.org/10.3390/ijms24032397.
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Microsorum scolopendria is an important medicinal plant that belongs to the Polypodiaceae family. In this study, we analyzed the effects of foliar spraying of chitosan on growth promotion and 20-hydroxyecdysone (20E) production in M. scolopendria. Treatment with chitosan at a concentration of 50 mg/L in both young and mature sterile fronds induced the highest increase in the amount of accumulated 20E. Using RNA sequencing, we identified 3552 differentially expressed genes (DEGs) in response to chitosan treatment. The identified DEGs were associated with 236 metabolic pathways. We identified several DEGs involved in the terpenoid and steroid biosynthetic pathways that might be associated with secondary metabolite 20E biosynthesis. Eight upregulated genes involved in cholesterol and phytosterol biosynthetic pathway, five upregulated genes related to the methylerythritol 4-phosphate (MEP) and mevalonate (MVA) pathways, and several DEGs that are members of cytochrome P450s and ABC transporters were identified. Quantitative real-time RT-PCR confirmed the results of RNA-sequencing. Taken together, we showed that chitosan treatment increased plant dry weight and 20E accumulation in M. scolopendria. RNA-sequencing and DEG analyses revealed key enzymes that might be related to the production of the secondary metabolite 20E in M. scolopendria.
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Mahadi, Nursyah Fitri, Azman Abd Samad, and Abdul Fatah A. Samad. "Identification of MiR398 and Its Regulatory Roles in Terpenoid Biosynthesis of Persicaria odorata." Malaysian Journal of Fundamental and Applied Sciences 20, no. 2 (April 24, 2024): 401–11. http://dx.doi.org/10.11113/mjfas.v20n2.3248.
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Persicaria odorata is an herbaceous plant with antifungal and antibacterial properties. The plant produces secondary metabolites, including phenols, sulphur-containing compounds and terpenoids, in response to biotic and abiotic stresses. Terpenoids in P. odorata are synthesized through the mevalonate (MVA) and the methylerythritol phosphate (MEP) pathways. 1-deoxy-D-xylulose-5phosphate isomerase (DXR) is a rate-limiting enzyme in the MEP pathway and may be regulated by microRNA (miRNA) miR398. There is a lack of evidence showing miR398 regulation in terpenoid biosynthesis in P. odorata through DXR-targeting. The study aimed to verify the stem-loop structure of miR398 and analyse its expression towards the target gene, DXR, in treated and control P. odorata. RNA was quantified and qualitatively analysed using a Nanodrop spectrophotometer and agarose gel electrophoresis. The stem loop of miR398 was verified using reverse transcriptase PCR (RT-PCR) and agarose gel electrophoresis. The expression of miR398 and DXR was compared between control and treated samples. Treated leaves were punctured with needles and left for 48h before harvest. Gene expressions were quantified and normalised using reference genes. The stem-loop structure of miR398 was confirmed, despite possible primer mismatches and unspecific binding. This step was essential before comparing and assessing the gene expression of miR398 and DXR. A decrease in abundance of miR398 whereas in increase in abundance was in the treated sample compared to the control sample indicating that miR398 negatively regulated DXR under stress conditions, suggesting an increase in terpenoid synthesis as a defence mechanism. DXR acts as a rate-limiting enzyme in the MEP metabolic pathway. Further studies are needed to quantify the effects of miR398 on terpenoid biosynthesis after wounding in P. odorata.
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Guggisberg, Ann M., Rachel E. Amthor, and Audrey R. Odom. "Isoprenoid Biosynthesis in Plasmodium falciparum." Eukaryotic Cell 13, no. 11 (September 12, 2014): 1348–59. http://dx.doi.org/10.1128/ec.00160-14.
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ABSTRACTMalaria kills nearly 1 million people each year, and the protozoan parasitePlasmodium falciparumhas become increasingly resistant to current therapies. Isoprenoid synthesis via the methylerythritol phosphate (MEP) pathway represents an attractive target for the development of new antimalarials. The phosphonic acid antibiotic fosmidomycin is a specific inhibitor of isoprenoid synthesis and has been a helpful tool to outline the essential functions of isoprenoid biosynthesis inP. falciparum. Isoprenoids are a large, diverse class of hydrocarbons that function in a variety of essential cellular processes in eukaryotes. InP. falciparum, isoprenoids are used for tRNA isopentenylation and protein prenylation, as well as the synthesis of vitamin E, carotenoids, ubiquinone, and dolichols. Recently, isoprenoid synthesis inP. falciparumhas been shown to be regulated by a sugar phosphatase. We outline what is known about isoprenoid function and the regulation of isoprenoid synthesis inP. falciparum, in order to identify valuable directions for future research.
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Chen, Cathy, Philip Frasse, Dana Hodge, Brianne Roper, and Audrey R. Odom John. "HAD2 Regulates Central Carbon Metabolism in Malaria Parasite P. falciparum." Journal of the Pediatric Infectious Diseases Society 12, Supplement_1 (November 1, 2023): S14. http://dx.doi.org/10.1093/jpids/piad070.026.
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Abstract Background The malaria parasite Plasmodium falciparum causes nearly half a million deaths each year. Widespread resistance to antimalarials prompts an urgent need for novel drug targets both unique and essential to the parasite. The methylerythritol (MEP) pathway of isoprenoid biosynthesis fits this profile. Previous studies showed that P. falciparum controls substrates available to the MEP pathway via the phosphatase HAD2. A point mutation in the HAD2 gene was found to misdirect metabolites from glycolysis to the MEP pathway. Elucidating the mechanism by which HAD2 controls central carbon metabolism will inform our understanding of essential parasite biology and the potential for resistance to metabolic inhibitors under development. Methods In this work, we have generated a P. falciparum strain in which HAD2 expression is under control of anhydrous tetracycline (aTc), such that growth in the absence of aTc leads to loss of HAD2 expression. We have used this tunable knockdown strain to regulate expression of HAD2 in asexual-stage parasites to test whether HAD2 is required for glycolytic and isoprenoid biosynthesis homeostasis. Asexual parasite growth rate was measured through flow cytometry, and dose-response assays were performed against a competitive small molecule inhibitor of the MEP pathway. Targeted metabolite profiling with liquid chromatography-tandem mass spectrometry (LC-MS/MS) is in process to quantify glycolytic and isoprenoid metabolites in this knockdown strain. Results Knockdown parasites off aTc exhibited reduced growth rates relative to the wild-type strain. These parasites also exhibited a 4-fold increase in resistance to a competitive inhibitor of the MEP pathway compared to the wild-type [0.755 +/- 0.12 µM vs. 3.201 +/- 0.35 µM; p<0.001). Conclusions Reduced expression of HAD2 confers resistance to a competitive inhibitor of the MEP pathway, consistent with the effect of elevated levels of MEP pathway intermediates as a result of disrupted homeostasis. Ongoing work will use this strain to understand the role of HAD2 on parasite metabolism, parasite growth, and drug resistance.
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Zhang, Yueya, Haifeng Yan, Yuan Li, Yuping Xiong, Meiyun Niu, Xinhua Zhang, Jaime A. Teixeira da Silva, and Guohua Ma. "Molecular Cloning and Functional Analysis of 1-Deoxy-D-Xylulose 5-Phosphate Reductoisomerase from Santalum album." Genes 12, no. 5 (April 22, 2021): 626. http://dx.doi.org/10.3390/genes12050626.
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Sandalwood (Santalum album L.) heartwood-derived essential oil contains a high content of sesquiterpenoids that are economically highly valued and widely used in the fragrance industry. Sesquiterpenoids are biosynthesized via the mevalonate acid and methylerythritol phosphate (MEP) pathways, which are also the sources of precursors for photosynthetic pigments. 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) is a secondary rate-limiting enzyme in the MEP pathway. In this paper, the 1416-bp open reading frame of SaDXR and its 897-bp promoter region, which contains putative conserved cis-elements involved in stress responsiveness (HSE and TC-rich repeats), hormone signaling (abscisic acid, gibberellin and salicylic acid) and light responsiveness, were cloned from 7-year-old S. album trees. A bioinformatics analysis suggested that SaDXR encodes a functional and conserved DXR protein. SaDXR was widely expressed in multiple tissues, including roots, twigs, stem sapwood, leaves, flowers, fruit and stem heartwood, displaying significantly higher levels in tissues with photosynthetic pigments, like twigs, leaves and flowers. SaDXR mRNA expression increased in etiolated seedlings exposed to light, and the content of chlorophylls and carotenoids was enhanced in all 35S::SaDXR transgenic Arabidopsis thaliana lines, consistent with the SaDXR expression level. SaDXR was also stimulated by MeJA and H2O2 in seedling roots. α-Santalol content decreased in response to fosmidomycin, a DXR inhibitor. These results suggest that SaDXR plays an important role in the biosynthesis of photosynthetic pigments, shifting the flux to sandalwood-specific sesquiterpenoids.
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Park, Jooyoung, Ann M. Guggisberg, Audrey R. Odom, and Niraj H. Tolia. "Cap-domain closure enables diverse substrate recognition by the C2-type haloacid dehalogenase-like sugar phosphatasePlasmodium falciparumHAD1." Acta Crystallographica Section D Biological Crystallography 71, no. 9 (August 25, 2015): 1824–34. http://dx.doi.org/10.1107/s1399004715012067.
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Haloacid dehalogenases (HADs) are a large enzyme superfamily of more than 500 000 members with roles in numerous metabolic pathways.Plasmodium falciparumHAD1 (PfHAD1) is a sugar phosphatase that regulates the methylerythritol phosphate (MEP) pathway for isoprenoid synthesis in malaria parasites. However, the structural determinants for diverse substrate recognition by HADs are unknown. Here, crystal structures were determined of PfHAD1 in complex with three sugar phosphates selected from a panel of diverse substrates that it utilizes. Cap-open and cap-closed conformations are observed, with cap closure facilitating substrate binding and ordering. These structural changes define the role of cap movement within the major subcategory of C2 HAD enzymes. The structures of an HAD bound to multiple substrates identifies binding and specificity-determining residues that define the structural basis for substrate recognition and catalysis within the HAD superfamily. While the substrate-binding region of the cap domain is flexible in the open conformations, this region becomes ordered and makes direct interactions with the substrate in the closed conformations. These studies further inform the structural and biochemical basis for catalysis within a large superfamily of HAD enzymes with diverse functions.