Overexpression of oilseed rape trehalose-6-phosphate synthesis gene BnaC02.TPS8 confers sensitivity to low nitrogen and high sucrose-induced anthocyanin accumulation in Arabidopsis.

MAIN CONCLUSION: Overexpression of BnaC02.TPS8 increased low N and high sucrose-induced anthocyanin accumulation. Anthocyanin plays a crucial role in safeguarding photosynthetic tissues against high light, UV radiation, and oxidative stress. Their accumulation is triggered by low nitrogen (N) stress and elevated sucrose levels in Arabidopsis. Trehalose-6-phosphate (T6P) serves as a pivotal signaling molecule, sensing sucrose availability, and carbon (C) metabolism. However, the mechanisms governing the regulation of T6P synthase (TPS) genes responsible for anthocyanin accumulation under conditions of low N and high sucrose remain elusive. In a previous study, we demonstrated the positive impact of a cytoplasm-localized class II TPS protein 'BnaC02.TPS8' on photosynthesis and seed yield improvement in Brassica napus. The present research delves into the biological role of BnaC02.TPS8 in response to low N and high sucrose. Ectopic overexpression of BnaC02.TPS8 in Arabidopsis seedlings resulted in elevated shoot T6P levels under N-sufficient conditions, as well as an increased carbon-to-nitrogen (C/N) ratio, sucrose accumulation, and starch storage under low N conditions. Overexpression of BnaC02.TPS8 in Arabidopsis heightened sensitivity to low N stress and high sucrose levels, accompanied by increased anthocyanin accumulation and upregulation of genes involved in flavonoid biosynthesis and regulation. Metabolic profiling revealed increased levels of intermediate products of carbon metabolism, as well as anthocyanin and flavonoid derivatives in BnaC02.TPS8-overexpressing Arabidopsis plants under low N conditions. Furthermore, yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) analyses demonstrated that BnaC02.TPS8 interacts with both BnaC08.TPS9 and BnaA01.TPS10. These findings contribute to our understanding of how TPS8-mediated anthocyanin accumulation is modulated under low N and high sucrose conditions.

Sucrose homeostasis: Mechanisms and opportunity in crop yield improvement.

Sugar homeostasis is a critical feature of biological systems. In humans, raised and dysregulated blood sugar is a serious health issue. In plants, directed changes in sucrose homeostasis and allocation represent opportunities in crop improvement. Plant tissue sucrose varies more than blood glucose and is found at higher concentrations (cytosol and phloem ca. 100 mM v 3.9-6.9 mM for blood glucose). Tissue sucrose varies with developmental stage and environment, but cytosol and phloem exhibit tight sucrose control. Sucrose homeostasis is a consequence of the integration of photosynthesis, synthesis of storage end-products such as starch, transport of sucrose to sinks and sink metabolism. Trehalose 6-phosphate (T6P)-SnRK1 and TOR play central, still emerging roles in regulating and coordinating these processes. Overall, tissue sucrose levels are more strongly related to growth than to photosynthesis. As a key sucrose signal, T6P regulates sucrose levels, transport and metabolic pathways to coordinate source and sink at a whole plant level. Emerging evidence shows that T6P interacts with meristems. With careful targeting, T6P manipulation through exploiting natural variation, chemical intervention and genetic modification is delivering benefits for crop yields. Regulation of cereal grain set, filling and retention may be the most strategically important aspect of sucrose allocation and homeostasis for food security.

Site-Selective Palladium-catalyzed Oxidation of Unprotected Aminoglycosides and Sugar Phosphates.

The site-selective modification of complex biomolecules by transition metal-catalysis is highly warranted, but often thwarted by the presence of Lewis basic functional groups. This study demonstrates that protonation of amines and phosphates in carbohydrates circumvents catalyst inhibition in palladium-catalyzed site-selective oxidation. Both aminoglycosides and sugar phosphates, compound classes that up till now largely escaped direct modification, are oxidized with good efficiency. Site-selective oxidation of kanamycin and amikacin was used to prepare a set of 3'-modified aminoglycoside derivatives of which two showed promising activity against antibiotic-resistant E. coli strains.

The Complex GH32 Enzyme Orchestra from Priestia megaterium Holds the Key to Better Discriminate Sucrose-6-phosphate Hydrolases from Other ß-Fructofuranosidases in Bacteria.

Inulin is widely used as a prebiotic and emerging as a priming compound to counteract plant diseases. We isolated inulin-degrading strains from the lettuce phyllosphere, identified as Bacillus subtilis and Priestia megaterium, species hosting well-known biocontrol organisms. To better understand their varying inulin degradation strategies, three intracellular ß-fructofuranosidases from P. megaterium NBRC15308 were characterized after expression in Escherichia coli: a predicted sucrose-6-phosphate (Suc6P) hydrolase (SacAP1, supported by molecular docking), an exofructanase (SacAP2), and an invertase (SacAP3). Based on protein multiple sequence and structure alignments of bacterial glycoside hydrolase family 32 enzymes, we identified conserved residues predicted to be involved in binding phosphorylated (Suc6P hydrolases) or nonphosphorylated substrates (invertases and fructanases). Suc6P hydrolases feature positively charged residues near the structural catalytic pocket (histidine, arginine, or lysine), whereas other ß-fructofuranosidases contain tryptophans. This correlates with our phylogenetic tree, grouping all predicted Suc6P hydrolases in a clan associated with genomic regions coding for transporters involved in substrate phosphorylation. These results will help to discriminate between Suc6P hydrolases and other ß-fructofuranosidases in future studies and to better understand the interaction of B. subtilis and P. megaterium endophytes with sucrose and/or fructans, sugars naturally present in plants or exogenously applied in the context of defense priming.

Cloning and Functional Characterization of 2-C-methyl-D-erythritol-4-phosphate cytidylyltransferase (LiMCT) Gene in Oriental Lily (Lilium 'Sorbonne').

2-C-methyl-D-erythritol-phosphate cytidylyltransferase (MCT) is a key enzyme in the MEP pathway of monoterpene synthesis, catalyzing the generation of 4- (5'-pyrophosphate cytidine)-2-C-methyl-D-erythritol from 2-C-methyl-D-erythritol-4-phosphate. We used homologous cloning strategy to clone gene, LiMCT, in the MEP pathway that may be involved in the regulation of floral fragrance synthesis in the Lilium oriental hybrid 'Sorbonne.' The full-length ORF sequence was 837 bp, encoding 278 amino acids. Bioinformatics analysis showed that the relative molecular weight of LiMCT protein is 68.56 kD and the isoelectric point (pI) is 5.12. The expression pattern of LiMCT gene was found to be consistent with the accumulation sites and emission patterns of floral fragrance monoterpenes in transcriptome data (unpublished). Subcellular localization indicated that the LiMCT protein is located in chloroplasts, which is consistent with the location of MEP pathway genes functioning in plastids to produce isoprene precursors. Overexpression of LiMCT in Arabidopsis thaliana affected the expression levels of MEP and MVA pathway genes, suggesting that overexpression of the LiMCT in A. thaliana affected the metabolic flow of C5 precursors of two different terpene synthesis pathways. The expression of the monoterpene synthase AtTPS14 was elevated nearly fourfold in transgenic A. thaliana compared with the control, and the levels of carotenoids and chlorophylls, the end products of the MEP pathway, were significantly increased in the leaves at full bloom, indicating that LiMCT plays an important role in regulating monoterpene synthesis and in the synthesis of other isoprene-like precursors in transgenic A. thaliana flowers. However, the specific mechanism of LiMCT in promoting the accumulation of isoprene products of the MEP pathway and the biosynthesis of floral monoterpene volatile components needs further investigation.

Design and synthesis of fosmidomycin analogs containing aza-linkers and their biological activity evaluation.

BACKGROUND: The enzymes involved in the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway are attractive targets of a new mode of action for developing anti-infective drugs and herbicides, and inhibitors against 1-deoxy-d-xylulose 5-phosphate reductoisomerase (IspC), the second key enzyme in the pathway, have been intensively investigated; however, few works are reported regarding IspC inhibitors designed for new herbicide discovery. RESULTS: A series of fosmidomycin (FOS) analogs were designed with nitrogen-containing linkers replacing the trimethylene linker between the two active substructures of FOS, phosphonic acid and hydroxamic acid. Synthesis followed a facile three-step route of sequential aza-Michael addition of α-amino acids to dibenzyl vinylphosphonate, amidation of the amino acid carboxyl with O-benzyl hydroxylamine, and simultaneous removal of the benzyl protective groups. Biological activity evaluation of IspC and model plants revealed that some compounds had moderate enzyme and model plant growth inhibition effects. In particular, compound 10g, which has a N-(4-fluorophenylethyl) nitrogen-containing linker, exhibited the best plant inhibition activities, superior to the control FOS against the model plants Arabidopsis thaliana, Brassica napus L., Amaranthus retroflexus and Echinochloa crus-galli. A dimethylallyl pyrophosphate rescue assay on A. thaliana confirmed that both 10g and FOS exert their herbicidal activity by blocking the MEP pathway. This result consistent with molecular docking, which confirmed 10g and FOS binding to the IspC active site in a similar way. CONCLUSION: Compound 10g has excellent herbicidal activity and represents the first herbicide lead structure of a new mode of action that targets IspC enzyme in the MEP pathway. © 2023 Society of Chemical Industry.

A versatile in situ cofactor enhancing system for meeting cellular demands for engineered metabolic pathways.

Cofactor imbalance obstructs the productivities of metabolically engineered cells. Herein, we employed a minimally perturbing system, xylose reductase and lactose (XR/lactose), to increase the levels of a pool of sugar phosphates which are connected to the biosynthesis of NAD(P)H, FAD, FMN, and ATP in Escherichia coli. The XR/lactose system could increase the amounts of the precursors of these cofactors and was tested with three different metabolically engineered cell systems (fatty alcohol biosynthesis, bioluminescence light generation, and alkane biosynthesis) with different cofactor demands. Productivities of these cells were increased 2-4-fold by the XR/lactose system. Untargeted metabolomic analysis revealed different metabolite patterns among these cells, demonstrating that only metabolites involved in relevant cofactor biosynthesis were altered. The results were also confirmed by transcriptomic analysis. Another sugar reducing system (glucose dehydrogenase) could also be used to increase fatty alcohol production but resulted in less yield enhancement than XR. This work demonstrates that the approach of increasing cellular sugar phosphates can be a generic tool to increase in vivo cofactor generation upon cellular demand for synthetic biology.

The trehalose 6-phosphate phosphatase family in plants.

Trehalose 6-phosphate (Tre6P), the intermediate of trehalose biosynthesis, is an essential signalling metabolite linking plant growth and development to carbon metabolism. While recent work has focused predominantly on the enzymes that produce Tre6P, little is known about the proteins that catalyse its degradation, the trehalose 6-phosphate phosphatases (TPPs). Often occurring in large protein families, TPPs exhibit cell-, tissue- and developmental stage-specific expression patterns, suggesting important regulatory functions in controlling local levels of Tre6P and trehalose as well as Tre6P signalling. Furthermore, growing evidence through gene expression studies and transgenic approaches shows that TPPs play an important role in integrating environmental signals with plant metabolism. This review highlights the large diversity of TPP isoforms in model and crop plants and identifies how modulating Tre6P metabolism in certain cell types, tissues, and at different developmental stages may promote stress tolerance, resilience and increased crop yield.

Functions of sucrose and trehalose 6-phosphate in controlling plant development.

Plants exhibit enormous plasticity in regulating their architecture to be able to adapt to a constantly changing environment and carry out vital functions such as photosynthesis, anchoring, and nutrient uptake. Phytohormones play a role in regulating these responses, but sugar signalling mechanisms are also crucial. Sucrose is not only an important source of carbon and energy fuelling plant growth, but it also functions as a signalling molecule that influences various developmental processes. Trehalose 6-phosphate (Tre6P), a sucrose-specific signalling metabolite, is emerging as an important regulator in plant metabolism and development. Key players involved in sucrose and Tre6P signalling pathways, including MAX2, SnRK1, bZIP11, and TOR, have been implicated in processes such as flowering, branching, and root growth. We will summarize our current knowledge of how these pathways shape shoot and root architecture and highlight how sucrose and Tre6P signalling are integrated with known signalling networks in shaping plant growth.

Carbohydrate-Templated Syntheses of Trifluoromethyl-Substituted MEP Analogues for the Study of the Methylerythritol Phosphate Pathway.

Trifluoromethyl analogues of methylerythritol phosphate (MEP) and 2-C-methyl-erythritol 2,4-cyclodiphosphate (MEcPP), natural substrates of key enzymes from the MEP pathway, were prepared starting from d-glucose as the chiral template to secure absolute configurations. The obligate trifluoromethyl group was inserted with complete diastereoselectivity using the Ruppert-Prakash nucleophile. Target compounds were assayed against the corresponding enzymes showing that trifluoro-MEP did not disrupt IspD activity, whereas trifluoro-MEcPP induced 40% inhibition of IspG at 1 mM.

Trehalose-6-phosphate signaling regulates lateral root formation in Arabidopsis thaliana.

Plant roots explore the soil for water and nutrients, thereby determining plant fitness and agricultural yield, as well as determining ground substructure, water levels, and global carbon sequestration. The colonization of the soil requires investment of carbon and energy, but how sugar and energy signaling are integrated with root branching is unknown. Here, we show through combined genetic and chemical modulation of signaling pathways that the sugar small-molecule signal, trehalose-6-phosphate (T6P) regulates root branching through master kinases SNF1-related kinase-1 (SnRK1) and Target of Rapamycin (TOR) and with the involvement of the plant hormone auxin. Increase of T6P levels both via genetic targeting in lateral root (LR) founder cells and through light-activated release of the presignaling T6P-precursor reveals that T6P increases root branching through coordinated inhibition of SnRK1 and activation of TOR. Auxin, the master regulator of LR formation, impacts this T6P function by transcriptionally down-regulating the T6P-degrader trehalose phosphate phosphatase B in LR cells. Our results reveal a regulatory energy-balance network for LR formation that links the 'sugar signal' T6P to both SnRK1 and TOR downstream of auxin.

Identification of levoglucosan degradation pathways in bacteria and sequence similarity network analysis.

Levoglucosan is produced in the pyrolysis of cellulose and starch, including from bushfires or the burning of biofuels, and is deposited from the atmosphere across the surface of the earth. We describe two levoglucosan degrading Paenarthrobacter spp. (Paenarthrobacter nitrojuajacolis LG01 and Paenarthrobacter histidinolovorans LG02) that were isolated from soil by metabolic enrichment using levoglucosan as the sole carbon source. Genome sequencing and proteomics analysis revealed the expression of a series of genes encoding known levoglucosan degrading enzymes, levoglucosan dehydrogenase (LGDH, LgdA), 3-keto-levoglucosan ß -eliminase (LgdB1) and glucose 3-dehydrogenase (LgdC), along with an ABC transporter cassette and an associated solute binding protein. However, no homologues of 3-ketoglucose dehydratase (LgdB2) were evident, while the expressed genes contained a range of putative sugar phosphate isomerases/xylose isomerases with weak similarity to LgdB2. Sequence similarity network analysis of genome neighbours of LgdA revealed that homologues of LgdB1 and LgdC are generally conserved in a range of bacteria in the phyla Firmicutes, Actinobacteria and Proteobacteria. One group of sugar phosphate isomerase/xylose isomerase homologues (named LgdB3) was identified with limited distribution that is mutually exclusive with LgdB2, and we propose that they may fulfil a similar function. LgdB1, LgdB2 and LgdB3 adopt similar predicted 3D folds, suggesting overlapping function in processing intermediates in LG metabolism. Our findings highlight diversity within the LGDH pathway, through which bacteria utilize levoglucosan as a nutrient source.

Not Every Hit-Identification Technique Works on 1-Deoxy-d-Xylulose 5-Phosphate Synthase (DXPS): Making the Most of a Virtual Screening Campaign.

In this work, we demonstrate how important it is to investigate not only on-target activity but to keep antibiotic activity against critical pathogens in mind. Since antimicrobial resistance is spreading in bacteria such as Mycobacterium tuberculosis, investigations into new targets are urgently needed. One promising new target is 1-deoxy-d-xylulose 5-phosphate synthase (DXPS) of the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway. We have recently solved the crystal structure of truncated M. tuberculosis DXPS and used it to perform a virtual screening in collaboration with Atomwise Inc. using their deep convolutional neural network-based AtomNet® platform. Of 94 virtual hit compounds only one showed interesting results in binding and activity studies. We synthesized 30 close derivatives using a straightforward synthetic route that allowed for easy derivatization. However, no improvement in activity was observed for any of the derivatives. Therefore, we tested them against a variety of pathogens and found them to be good inhibitors against Escherichia coli.

Liquid chromatography-mass spectrometry analyses of polyprenyl phosphates in Escherichia coli cells in which genes for isoprenoid synthesis are amplified.

Overproduction of isopentenyl diphosphate by the amplification of the genes for the methylerythritol 4-phosphate pathway, dxs and dxr, is known to be deleterious for the growth of Escherichia coli. We hypothesized that overproduction of one of the endogenous isoprenoids, in addition to isopentenyl diphosphate itself, might be the cause of the reported reduced growth rate and attempted to identify the causative agent. In order to analyze polyprenyl phosphates, they were methylated by the reaction with diazomethane. The resulting dimethyl esters of polyprenyl phosphates with carbon numbers from 40 to 60 were quantitated by high-performance liquid chromatography-mass spectrometric analysis detecting ion peaks of the sodium ion adducts. The E. coli was transformed by a multi-copy plasmid carrying both the dxs and dxr genes. Amplification of dxs and dxr significantly increased the levels of polyprenyl phosphates and 2-octaprenylphenol. The levels of Z,E-mixed polyprenyl phosphates with carbon numbers of 50-60 in the strain in which ispB was co-amplified with dxs and dxr were lower than those in the control strain where only dxs and dxr were amplified. The levels of (all-E)-octaprenyl phosphate and 2-octaprenylphenol in the strains in which ispU/rth or crtE was co-amplified with dxs and dxr were lower than those in the control strain. Although the increase in the level of each isoprenoid intermediate was blocked, the growth rates of these strains were not restored. Neither polyprenyl phosphates nor 2-octaprenylphenol can be determined to be the cause of the growth rate reduction seen with dxs and dxr amplification.

The Multifaceted MEP Pathway: Towards New Therapeutic Perspectives.

Isoprenoids, a diverse class of natural products, are present in all living organisms. Their two universal building blocks are synthesized via two independent pathways: the mevalonate pathway and the 2-C-methyl-ᴅ-erythritol 4-phosphate (MEP) pathway. The presence of the latter in pathogenic bacteria and its absence in humans make all its enzymes suitable targets for the development of novel antibacterial drugs. (E)-4-Hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP), the last intermediate of this pathway, is a natural ligand for the human Vγ9Vδ2 T cells and the most potent natural phosphoantigen known to date. Moreover, 5-hydroxypentane-2,3-dione, a metabolite produced by Escherichia coli 1-deoxy-ᴅ-xylulose 5-phosphate synthase (DXS), the first enzyme of the MEP pathway, structurally resembles (S)-4,5-dihydroxy-2,3-pentanedione, a signal molecule implied in bacterial cell communication. In this review, we shed light on the diversity of potential uses of the MEP pathway in antibacterial therapies, starting with an overview of the antibacterials developed for each of its enzymes. Then, we provide insight into HMBPP, its synthetic analogs, and their prodrugs. Finally, we discuss the potential contribution of the MEP pathway to quorum sensing mechanisms. The MEP pathway, providing simultaneously antibacterial drug targets and potent immunostimulants, coupled with its potential role in bacterial cell-cell communication, opens new therapeutic perspectives.

Isomer selectivity of one- and two-dimensional approaches of mixed-mode and hydrophilic interaction liquid chromatography coupled to tandem mass spectrometry for sugar phosphates of glycolysis and pentose phosphate pathways.

In this study, the chromatographic behavior of mixed-mode and hydrophilic interaction liquid chromatography (HILIC) with the mixed-mode HILIC/strong anion-exchange (SAX) column HILICpak VT-50 2D and the two HILIC columns Atlantis Premier BEH Z-HILIC and Acquity Premier BEH Amide was assessed with regard to their separation capability of the metabolites from the glycolysis and pentose phosphate pathways. Chromatographic conditions were evaluated with the aim of achieving separation of the isomeric glycolytic phosphorylated carbohydrate metabolites free from isomeric interferences and thus allowing for selective targeted analysis by liquid chromatography with tandem mass spectrometry (MS/MS) using multiple reaction monitoring acquisition. The effects of pH values (8.0/9.0/10.0) of the ammonium bicarbonate buffer and gradient time were investigated during HILIC-MS/MS analysis, with the optimal conditions found at pH = 10.0. Separation of the pentose phosphate isomers (ribose 5- and 1-phosphate, xylulose 5-phosphate and ribulose 5-phosphate) was achieved on the mixed-mode HILIC/SAX (HILICpak VT-50 2D) column and HILIC BEH Amide column. Column performance was evaluated based on the direct comparison of chromatographic parameters, i.e. peak width at 50% and peak tailing factors of the individual metabolites. Parity plots were generated allowing a direct comparison between the normalized retention times and assessment of orthogonality of all 3 stationary phases evaluated. Separation of 7 biologically relevant hexose monophosphates metabolites turned out to be challenging by HILIC-MS/MS, with the BEH Amide providing the best individual results for such a separation. However, fructose 6-phosphate and glucose 1-phosphate co-eluted. Therefore, an on-line heart-cutting HILIC-Mixed Mode 2D-LC-QToF experiment was conducted, allowing the separation of this critical isomer pair. In this setup, the BEH Amide column in the 1D separated the majority of target metabolites, while a heart-cut of the peak from totally coeluted fructose 6-phosphate and glucose 1-phosphate was separated in the 2D with HILICpak VT50-2D column, thus allowing undisturbed determination of the glycolytic phosphorylated carbohydrate metabolites due to their chromatographic separation from hexose monophosphate metabolites. The assay specificity towards 7 common hexose monophosphates was characterized (glucose 1- and 6-phosphate, galactose 1- and 6-phosphate, fructose 6-phosphate, mannose 1- and 6-phosphate). The selectivity of some rare hexose monophosphates (allose 6-phosphate, tagatose 6-phosphate, sorbose 1-phosphate) was also tested.

Using phosphoglucose isomerase-deficient (pgi1Δ) Saccharomyces cerevisiae to map the impact of sugar phosphate levels on D-glucose and D-xylose sensing.

BACKGROUND: Despite decades of engineering efforts, recombinant Saccharomyces cerevisiae are still less efficient at converting D-xylose sugar to ethanol compared to the preferred sugar D-glucose. Using GFP-based biosensors reporting for the three main sugar sensing routes, we recently demonstrated that the sensing response to high concentrations of D-xylose is similar to the response seen on low concentrations of D-glucose. The formation of glycolytic intermediates was hypothesized to be a potential cause of this sensing response. In order to investigate this, glycolysis was disrupted via the deletion of the phosphoglucose isomerase gene (PGI1) while intracellular sugar phosphate levels were monitored using a targeted metabolomic approach. Furthermore, the sugar sensing of the PGI1 deletants was compared to the PGI1-wildtype strains in the presence of various types and combinations of sugars. RESULTS: Metabolomic analysis revealed systemic changes in intracellular sugar phosphate levels after deletion of PGI1, with the expected accumulation of intermediates upstream of the Pgi1p reaction on D-glucose and downstream intermediates on D-xylose. Moreover, the analysis revealed a preferential formation of D-fructose-6-phosphate from D-xylose, as opposed to the accumulation of D-fructose-1,6-bisphosphate that is normally observed when PGI1 deletants are incubated on D-fructose. This may indicate a role of PFK27 in D-xylose sensing and utilization. Overall, the sensing response was different for the PGI1 deletants, and responses to sugars that enter the glycolysis upstream of Pgi1p (D-glucose and D-galactose) were more affected than the response to those entering downstream of the reaction (D-fructose and D-xylose). Furthermore, the simultaneous exposure to sugars that entered upstream and downstream of Pgi1p (D-glucose with D-fructose, or D-glucose with D-xylose) resulted in apparent synergetic activation and deactivation of the Snf3p/Rgt2p and cAMP/PKA pathways, respectively. CONCLUSIONS: Overall, the sensing assays indicated that the previously observed D-xylose response stems from the formation of downstream metabolic intermediates. Furthermore, our results indicate that the metabolic node around Pgi1p and the level of D-fructose-6-phosphate could represent attractive engineering targets for improved D-xylose utilization.

Mapping of the plant SnRK1 kinase signalling network reveals a key regulatory role for the class II T6P synthase-like proteins.

The central metabolic regulator SnRK1 controls plant growth and survival upon activation by energy depletion, but detailed molecular insight into its regulation and downstream targets is limited. Here we used phosphoproteomics to infer the sucrose-dependent processes targeted upon starvation by kinases as SnRK1, corroborating the relation of SnRK1 with metabolic enzymes and transcriptional regulators, while also pointing to SnRK1 control of intracellular trafficking. Next, we integrated affinity purification, proximity labelling and crosslinking mass spectrometry to map the protein interaction landscape, composition and structure of the SnRK1 heterotrimer, providing insight in its plant-specific regulation. At the intersection of this multi-dimensional interactome, we discovered a strong association of SnRK1 with class II T6P synthase (TPS)-like proteins. Biochemical and cellular assays show that TPS-like proteins function as negative regulators of SnRK1. Next to stable interactions with the TPS-like proteins, similar intricate connections were found with known regulators, suggesting that plants utilize an extended kinase complex to fine-tune SnRK1 activity for optimal responses to metabolic stress.

Targeted analysis of sugar phosphates from glycolysis pathway by phosphate methylation with liquid chromatography coupled to tandem mass spectrometry.

Monitoring the glycolysis pathway remains an analytical challenge as most metabolites involved are sugar phosphates. Structural similarity, instability, high polarity, and rich negative charges of sugar phosphates make LC-MS based analysis challenging. Here, we developed an improved workflow integrating uniformly 13C-labeled yeast metabolite extract, TiO2-based enrichment, differential stable isotope labeling phosphate methylation, porous graphic carbon column, and selected reaction monitoring acquisition. Uniformly 13C labeled yeast metabolite extract was used as internal standards while differential stable isotope labeled sugar phosphates worked as calibrants. The established method was validated in human plasma, platelet and cultured HeLa cells. The limits of quantification ranged between 0.25 and 0.54 pmol on column. The method was adapted and its applicability tested for human platelets in which activation with collagen-related peptide (CRP) clearly showed the upregulation of some SPx metabolites. The results document that this newly established method can be successfully used to monitor glycolysis in different biological samples. As an extension, more phosphorylated and carboxylated metabolites from the central carbon metabolism (pentose phosphate cycle, TCA cycle) were tested as well. This method showed superior performance, especially for multiple phosphorylated and carboxylated metabolites. For quantitative purpose, the concept of SPx in three sets (12C-analytes, U-13C-IS, deuterated calibrants) has the potential to be adapted for more anionic metabolites.

Hexokinase 2: The preferential target of trehalose-6-phosphate over hexokinase 1.

Cancer-related metabolic features are in part maintained by hexokinase 2 upregulation, which leads to high levels of glucose-6-phosphate (G6P) and is needed to provide energy and biomass to support rapid proliferation. Using a humanized model of the yeast Saccharomyces cerevisiae, we explored how human hexokinase 2 (HK2) behaves under different nutritional conditions. At high glucose levels, yeast presents aerobic glycolysis through a regulatory mechanism known as catabolic repression, which exerts a metabolic adaptation like the Warburg effect. At high glucose concentrations, HK2 did not translocate into the nucleus and was not able to shift the metabolism toward a highly glycolytic state, in contrast to the effect of yeast hexokinase 2 (Hxk2), which is a crucial protein for the control of aerobic glycolysis in S. cerevisiae. During the stationary phase, when glucose is exhausted, Hxk2 is shuttled out of the nucleus, ceasing catabolic repression. Cells harvested at this condition display low glucose consumption rates. However, glucose-starved cells expressing HK2 had an increased capacity to consume glucose. In those cells, HK2 localized to mitochondria, becoming insensitive to G6P inhibition. We also found that the sugar trehalose-6-phosphate (T6P) is a human HK2 inhibitor, like yeast Hxk2, but was not able to inhibit human HK1, the isoform that is ubiquitously expressed in almost all mammalian tissues. In contrast to G6P, T6P inhibited HK2 even when HK2 was associated with mitochondria. The binding of HK2 to mitochondria is crucial for cancer survival and proliferation. T6P was able to reduce the cell viability of tumor cells, although its toxicity was not impressive. This was expected as cell absorption of phosphorylated sugars is low, which might be counteracted using nanotechnology. Altogether, these data suggest that T6P may offer a new paradigm for cancer treatment based on specific inhibition of HK2.