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Microorganisms Mar 2022Hyperoside (quercetin 3--galactoside) exhibits many biological functions, along with higher bioactivities than quercetin. In this study, three UDP-dependent...
Hyperoside (quercetin 3--galactoside) exhibits many biological functions, along with higher bioactivities than quercetin. In this study, three UDP-dependent glycosyltransferases (UGTs) were screened for efficient hyperoside synthesis from quercetin. The highest hyperoside production of 58.5 mg·L was obtained in a recombinant co-expressing UGT from (PhUGT) and UDP-glucose epimerase (GalE, a key enzyme catalyzing the conversion of UDP-glucose to UDP-galactose) from . When additional enzymes (phosphoglucomutase (Pgm) and UDP-glucose pyrophosphorylase (GalU)) were introduced into the recombinant , the increased flux toward UDP-glucose synthesis led to enhanced UDP-galactose-derived hyperoside synthesis. The efficiency of the recombinant strain was further improved by increasing the copy number of the PhUGT, which is a limiting step in the bioconversion. Through the optimization of the fermentation conditions, the production of hyperoside increased from 245.6 to 411.2 mg·L. The production was also conducted using a substrate-fed batch fermentation, and the maximal hyperoside production was 831.6 mg·L, with a molar conversion ratio of 90.2% and a specific productivity of 27.7 mg·L·h after 30 h of fermentation. The efficient hyperoside synthesis pathway described here can be used widely for the glycosylation of other flavonoids and bioactive substances.
PubMed: 35336203
DOI: 10.3390/microorganisms10030628 -
Current Biology : CB Aug 2022All photosynthetic organisms balance CO assimilation with growth and carbon storage. Stored carbon is used for growth at night and when demand exceeds assimilation....
All photosynthetic organisms balance CO assimilation with growth and carbon storage. Stored carbon is used for growth at night and when demand exceeds assimilation. Gaining a mechanistic understanding of carbon partitioning between storage and growth in trees is important for biological studies and for estimating the potential of terrestrial photosynthesis to sequester anthropogenic CO emissions. Starch represents the main carbon storage in plants. To examine the carbon storage mechanism and role of starch during tree growth, we generated and characterized low-starch hybrid aspen (Populus tremula × tremuloides) trees using CRISPR-Cas9-mediated gene editing of two PHOSPHOGLUCOMUTASE (PGM) genes coding for plastidial PGM isoforms essential for starch biosynthesis. We demonstrate that starch deficiency does not reduce tree growth even in short days, showing that starch is not a critical carbon reserve during diel growth of aspen. The low-starch trees assimilated up to ∼30% less CO compared to the wild type under a range of irradiance levels, but this did not reduce growth or wood density. This implies that aspen growth is not limited by carbon assimilation under benign growth conditions. Moreover, the timing of bud set and bud flush in the low-starch trees was not altered, implying that starch reserves are not critical for the seasonal growth-dormancy cycle. The findings are consistent with a passive starch storage mechanism that contrasts with the annual Arabidopsis and indicate that the capacity of the aspen to absorb CO is limited by the rate of sink tissue growth.
Topics: Arabidopsis; Carbon; Carbon Dioxide; Photosynthesis; Plant Leaves; Populus; Starch
PubMed: 35820419
DOI: 10.1016/j.cub.2022.06.056 -
Experimental Biology and Medicine... Sep 2022The hexosamine biosynthetic pathway (HBP) is connected to abnormal N- and O-linked protein glycosylation in cancer, which performs critical roles in tumorigenesis....
The hexosamine biosynthetic pathway (HBP) is connected to abnormal N- and O-linked protein glycosylation in cancer, which performs critical roles in tumorigenesis. However, the regulation mechanisms of HBP and its role in colorectal cancer (CRC) progression remain unexplained. This study analyzed the expression level of phosphoglucomutase 3 (PGM3), a key enzyme in HBP, and identified its function in CRC cell lines. Analysis of publicly available CRC microarray data determined that PGM3 is upregulated in CRC tumor tissues. Furthermore, functional experiments emphasized the significant roles of PGM3 in facilitating CRC cell proliferation and migration. Mechanistically, we demonstrated that the activity of β-catenin in CRC was maintained by PGM3-mediated O-GlcNAcylation. PGM3 knockdown or inhibition of O-GlcNAc transferase decreased β-catenin activity and the expression levels of its downstream targets. Collectively, our findings indicate that PGM3 exhibits tumor-promoting roles by elevating O-GlcNAcylation level and maintaining β-catenin activity, and might serve as a prognostic biomarker and treatment target in CRC.
Topics: Biomarkers; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Transformation, Neoplastic; Colorectal Neoplasms; Gene Expression Regulation, Neoplastic; Hexosamines; Humans; Phosphoglucomutase; Wnt Signaling Pathway; beta Catenin
PubMed: 35723049
DOI: 10.1177/15353702221101810 -
Frontiers in Cellular and Infection... 2022The obligate intracellular bacteria store glycogen in the lumen of the vacuoles in which they grow. Glycogen catabolism generates glucose-1-phosphate (Glc1P), while the...
The obligate intracellular bacteria store glycogen in the lumen of the vacuoles in which they grow. Glycogen catabolism generates glucose-1-phosphate (Glc1P), while the bacteria can take up only glucose-6-phosphate (Glc6P). We tested whether the conversion of Glc1P into Glc6P could be catalyzed by a phosphoglucomutase (PGM) of host or bacterial origin. We found no evidence for the presence of the host PGM in the vacuole. Two proteins, CT295 and CT815, are potential PGMs. By reconstituting the reaction using purified proteins, and by complementing PGM deficient fibroblasts, we demonstrated that only CT295 displayed robust PGM activity. Intriguingly, we showed that glycogen accumulation in the lumen of the vacuole of a subset of species (, , ) correlated with the presence, in CT295 orthologs, of a secretion signal recognized by the type three secretion (T3S) machinery of . and do not accumulate glycogen, and their CT295 orthologs lack T3S signals. In conclusion, we established that the conversion of Glc1P into Glc6P was accomplished by a bacterial PGM, through the acquisition of a T3S signal in a "housekeeping" protein. Acquisition of this signal likely contributed to shaping glycogen metabolism within .
Topics: Chlamydia trachomatis; Glucose-6-Phosphate; Glycogen; Phosphoglucomutase; Vacuoles
PubMed: 35795184
DOI: 10.3389/fcimb.2022.866729 -
Yeast (Chichester, England) Jan 2021In many yeast species, the three genes at the centre of the galactose catabolism pathway, GAL1, GAL10 and GAL7, are neighbours in the genome and form a metabolic gene...
In many yeast species, the three genes at the centre of the galactose catabolism pathway, GAL1, GAL10 and GAL7, are neighbours in the genome and form a metabolic gene cluster. We report here that some yeast strains in the genus Torulaspora have much larger GAL clusters that include genes for melibiase (MEL1), galactose permease (GAL2), glucose transporter (HGT1), phosphoglucomutase (PGM1) and the transcription factor GAL4, in addition to GAL1, GAL10, and GAL7. Together, these eight genes encode almost all the steps in the pathway for catabolism of extracellular melibiose (a disaccharide of galactose and glucose). We show that a progenitor 5-gene cluster containing GAL 7-1-10-4-2 was likely present in the common ancestor of Torulaspora and Zygotorulaspora. It added PGM1 and MEL1 in the ancestor of most Torulaspora species. It underwent further expansion in the T. pretoriensis clade, involving the fusion of three progenitor clusters in tandem and the gain of HGT1. These giant GAL clusters are highly polymorphic in structure, and subject to horizontal transfers, pseudogenization and gene losses. We identify recent horizontal transfers of complete GAL clusters from T. franciscae into one strain of T. delbrueckii, and from a relative of T. maleeae into one strain of T. globosa. The variability and dynamic evolution of GAL clusters in Torulaspora indicates that there is strong natural selection on the GAL pathway in this genus.
Topics: Galactose; Genes, Fungal; Melibiose; Metabolic Networks and Pathways; Multigene Family; Torulaspora
PubMed: 33141945
DOI: 10.1002/yea.3532 -
International Journal of Molecular... Dec 2023Exogenous nitrogen and carbon can affect plant cell walls, which are composed of structural carbon. Sucrose synthase (SUS), invertase (INV), hexokinase (HXK),...
Exogenous nitrogen and carbon can affect plant cell walls, which are composed of structural carbon. Sucrose synthase (SUS), invertase (INV), hexokinase (HXK), phosphoglucomutase (PGM), and UDP-glucose pyrophosphorylase (UGP) are the key enzymes of sucrose metabolism involved in cell wall synthesis. To understand whether these genes are regulated by carbon and nitrogen to participate in structural carbon biosynthesis, we performed genome-wide identification, analyzed their expression patterns under different carbon and nitrogen treatments, and conducted preliminary functional verification. Different concentrations of nitrogen and carbon were applied to poplar ( Torr. and Gray), which caused changes in cellulose, lignin, and hemicellulose contents. In poplar, 6 s, 20 s, 6 s, 4 s, and 2 s were identified. Moreover, the physicochemical properties, collinearity, and tissue specificity were analyzed. The correlation analysis showed that the expression levels of /, ////, , , /, /, and were positively correlated with the cellulose content. Meanwhile, the knockout of significantly reduced the cellulose content. This study could lay the foundation for revealing the functions of s, s, s, , and s, which affected structural carbon synthesis regulated by nitrogen and carbon, proving that is involved in cell wall synthesis.
Topics: Populus; Cellulose; Lignin; Carbon; Nitrogen; Gene Expression Regulation, Plant
PubMed: 38139109
DOI: 10.3390/ijms242417277 -
BMC Research Notes May 2022Toxoplasma gondii is a ubiquitous parasite of medical and veterinary importance; however, there exists no cure for chronic toxoplasmosis. Metabolic enzymes required for...
OBJECTIVE
Toxoplasma gondii is a ubiquitous parasite of medical and veterinary importance; however, there exists no cure for chronic toxoplasmosis. Metabolic enzymes required for the production and maintenance of tissue cysts represent promising targets for novel therapies. Here, we use reverse genetics to investigate the role of Toxoplasma phosphoglucomutase 1, PGM1, in Toxoplasma growth and cystogenesis.
RESULTS
We found that disruption of pgm1 did not significantly affect Toxoplasma intracellular growth and the lytic cycle. pgm1-defective parasites could differentiate into bradyzoites and produced cysts containing amylopectin in vitro. However, cysts produced in the absence of pgm1 were significantly smaller than wildtype. Together, our findings suggest that PGM1 is dispensable for in vitro growth but contributes to optimal Toxoplasma cyst development in vitro, thereby necessitating further investigation into the function of this enzyme in Toxoplasma persistence in its host.
Topics: Humans; Phosphoglucomutase; Toxoplasma; Toxoplasmosis
PubMed: 35597992
DOI: 10.1186/s13104-022-06073-5 -
Journal of the American Chemical Society Feb 2021The activation barriers Δ for / for the reactions of whole substrates catalyzed by 6-phosphogluconate dehydrogenase, glucose 6-phosphate dehydrogenase, and glucose...
The activation barriers Δ for / for the reactions of whole substrates catalyzed by 6-phosphogluconate dehydrogenase, glucose 6-phosphate dehydrogenase, and glucose 6-phosphate isomerase are reduced by 11-13 kcal/mol by interactions between the protein and the substrate phosphodianion. Between 4 and 6 kcal/mol of this dianion binding energy is expressed at the transition state for phosphite dianion activation of the respective enzyme-catalyzed reactions of truncated substrates d-xylonate or d-xylose. These and earlier results from studies on β-phosphoglucomutase, triosephosphate isomerase, and glycerol 3-phosphate dehydrogenase define a cluster of six enzymes that catalyze reactions in glycolysis or of glycolytic intermediates, and which utilize substrate dianion binding energy for enzyme activation. Dianion-driven conformational changes, which convert flexible open proteins to tight protein cages for the phosphorylated substrate, have been thoroughly documented for five of these six enzymes. The clustering of metabolic enzymes which couple phosphodianion-driven conformational changes to enzyme activation suggests that this catalytic motif has been widely propagated in the proteome.
Topics: Biocatalysis; Enzyme Activation; Glucose-6-Phosphate Isomerase; Glucosephosphate Dehydrogenase; Kinetics; Phosphites; Phosphogluconate Dehydrogenase; Substrate Specificity; Thermodynamics; Xylose
PubMed: 33560827
DOI: 10.1021/jacs.0c13423 -
Biomolecular NMR Assignments Oct 2019β-Phosphoglucomutase (βPGM) is a magnesium-dependent phosphoryl transfer enzyme that catalyses the reversible isomerisation of β-glucose 1-phosphate and glucose...
β-Phosphoglucomutase (βPGM) is a magnesium-dependent phosphoryl transfer enzyme that catalyses the reversible isomerisation of β-glucose 1-phosphate and glucose 6-phosphate, via two phosphoryl transfer steps and a β-glucose 1,6-bisphosphate intermediate. Substrate-free βPGM is an essential component of the catalytic cycle and an understanding of its dynamics would present significant insights into βPGM functionality, and enzyme catalysed phosphoryl transfer in general. Previously, 30 residues around the active site of substrate-free βPGM were identified as undergoing extensive millisecond dynamics and were unassignable. Here we report H, N and C backbone resonance assignments of the P146A variant (βPGM) in its substrate-free form, where the K145-A146 peptide bond adopts a trans conformation in contrast to all crystal structures of βPGM, where the K145-P146 peptide bond is cis. In βPGM millisecond dynamics are suppressed for all but 17 residues, allowing 92% of backbone resonances to be assigned. Secondary structure predictions using TALOS-N reflect βPGM crystal structures, and a chemical shift comparison between substrate-free βPGM and βPGM confirms that the solution conformations are very similar, except for the D137-A147 loop. Hence, the isomerisation state of the 145-146 peptide bond has little effect on structure but the cis conformation triggers millisecond dynamics in the hinge (V12-T16), the nucleophile (D8) and residues that coordinate the transferring phosphate group (D8 and S114-S116), and the D137-A147 loop (V141-A142 and K145). These millisecond dynamics occur in addition to those for residues involved in coordinating the catalytic Mg ion and the L44-L53 loop responsible for substrate discrimination.
Topics: Lactococcus lactis; Mutant Proteins; Nuclear Magnetic Resonance, Biomolecular; Phosphoglucomutase
PubMed: 31396843
DOI: 10.1007/s12104-019-09904-y -
Journal of Experimental Botany Mar 2023Glycogen and starch are the main storage polysaccharides, acting as a source of carbon and energy when necessary. Interconversion of glucose-1-phosphate and...
Glycogen and starch are the main storage polysaccharides, acting as a source of carbon and energy when necessary. Interconversion of glucose-1-phosphate and glucose-6-phosphate by phosphoglucomutases connects the metabolism of these polysaccharides with central carbon metabolism. However, knowledge about how this connection affects the ability of cells to cope with environmental stresses is still scarce. The cyanobacterium Synechocystis sp. PCC 6803 has two enzymes with phosphoglucomutase activity, PGM (phosphoglucomutase) and PMM/PGM (phosphomannomutase/phosphoglucomutase). In this work, we generated a null mutant of PGM (∆PGM) that exhibits very reduced phosphoglucomutase activity (1% of wild type activity). Although this mutant accumulates moderate amounts of glycogen, its phenotype resembles that of glycogen-less mutants, including high light sensitivity and altered response to nitrogen deprivation. Using an on/off arsenite promoter, we demonstrate that PMM/PGM is essential for growth and responsible for the remaining phosphoglucomutase activity in the ∆PGM strain. Furthermore, overexpression of PMM/PGM in the ∆PGM strain is enough to revoke the phenotype of this mutant. These results emphasize the importance of an adequate flux between glycogen and central carbon metabolism to maintain cellular fitness and indicate that although PGM is the main phosphoglucomutase activity, the phosphoglucomutase activity of PMM/PGM can substitute it when expressed in sufficient amounts.
Topics: Phosphoglucomutase; Glycogen; Carbon; Starch; Cyanobacteria
PubMed: 36454663
DOI: 10.1093/jxb/erac474