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Biodesign Research 2022Maltose is a natural -(1,4)-linked disaccharide with wide applications in food industries and microbial fermentation. However, maltose has scarcely been used for...
Maltose is a natural -(1,4)-linked disaccharide with wide applications in food industries and microbial fermentation. However, maltose has scarcely been used for biosynthesis, possibly because its phosphorylation by maltose phosphorylase (MP) yields -glucose 1-phosphate (-G1P) that cannot be utilized by -phosphoglucomutase (-PGM) commonly found in synthetic enzymatic biosystems previously constructed by our group. Herein, we designed an synthetic enzymatic reaction module comprised of MP, -phosphoglucomutase (-PGM), and polyphosphate glucokinase (PPGK) for the stoichiometric conversion of each maltose molecule to two glucose 6-phosphate (G6P) molecules. Based on this synthetic module, we further constructed two synthetic biosystems to produce bioelectricity and fructose 1,6-diphosphate (FDP), respectively. The 14-enzyme biobattery achieved a Faraday efficiency of 96.4% and a maximal power density of 0.6 mW/cm, whereas the 5-enzyme FDP-producing biosystem yielded 187.0 mM FDP from 50 g/L (139 mM) maltose by adopting a fed-batch substrate feeding strategy. Our study not only suggests new application scenarios for maltose but also provides novel strategies for the high-efficient production of bioelectricity and value-added biochemicals.
PubMed: 37850132
DOI: 10.34133/2022/9806749 -
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 -
ACS Catalysis Mar 2022Understanding the factors that underpin the enormous catalytic proficiencies of enzymes is fundamental to catalysis and enzyme design. Enzymes are, in part, able to...
Understanding the factors that underpin the enormous catalytic proficiencies of enzymes is fundamental to catalysis and enzyme design. Enzymes are, in part, able to achieve high catalytic proficiencies by utilizing the binding energy derived from nonreacting portions of the substrate. In particular, enzymes with substrates containing a nonreacting phosphodianion group coordinated in a distal site have been suggested to exploit this binding energy primarily to facilitate a conformational change from an open inactive form to a closed active form, rather than to either induce ground state destabilization or stabilize the transition state. However, detailed structural evidence for the model is limited. Here, we use β-phosphoglucomutase (βPGM) to investigate the relationship between binding a phosphodianion group in a distal site, the adoption of a closed enzyme form, and catalytic proficiency. βPGM catalyzes the isomerization of β-glucose 1-phosphate to glucose 6-phosphate via phosphoryl transfer reactions in the proximal site, while coordinating a phosphodianion group of the substrate(s) in a distal site. βPGM has one of the largest catalytic proficiencies measured and undergoes significant domain closure during its catalytic cycle. We find that side chain substitution at the distal site results in decreased substrate binding that destabilizes the closed active form but is not sufficient to preclude the adoption of a fully closed, near-transition state conformation. Furthermore, we reveal that binding of a phosphodianion group in the distal site stimulates domain closure even in the absence of a transferring phosphoryl group in the proximal site, explaining the previously reported β-glucose 1-phosphate inhibition. Finally, our results support a trend whereby enzymes with high catalytic proficiencies involving phosphorylated substrates exhibit a greater requirement to stabilize the closed active form.
PubMed: 35692864
DOI: 10.1021/acscatal.1c05524 -
Cancer Cell International May 2022Phosphoglucomutase 1 (PGM1) is known for its involvement in cancer pathogenesis. However, its biological role in colorectal cancer (CRC) has remained unknown. Here, we...
BACKGROUND
Phosphoglucomutase 1 (PGM1) is known for its involvement in cancer pathogenesis. However, its biological role in colorectal cancer (CRC) has remained unknown. Here, we studied the functions and mechanisms of PGM1 in CRC.
METHODS
We verified PGM-1 as a differentially expressed gene (DEG) by employing a comprehensive strategy of TCGA-COAD dataset mining and computational biology. Relative levels of PGM-1 in CRC tumors and adjoining peritumoral tissues were determined by qRT-PCR, western blotting (WB), and immunohistochemical (IHC) staining in a tissue microarray. PGM1 functions were analyzed by CCK8, EdU, colony formation, cell cycle, apoptosis, and Transwell migration and invasion assays. The influence of PGM1 was further investigated by studying tumor formation in vivo.
RESULTS
The levels of PGM1 mRNA and protein were both reduced in CRC tissues, and the reductions were related to CRC pathology and overall survival. PGM1 knockdown stimulated both cell proliferation and colony formation, and inhibited cell cycle arrest and apoptosis, while overexpression of PGM1 produced the opposite effects in CRC cells both in vivo and in vitro. Furthermore, the effects of PGM1 were related to the PI3K/ AKT pathway.
CONCLUSION
We verified that PGM1 suppresses CRC progression via the PI3K/AKT pathway. These results suggest the potential for targeting PGM1 in treatment of CRC.
PubMed: 35614441
DOI: 10.1186/s12935-022-02545-7 -
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 -
Advanced Biomedical Research 2023Due to the increasing prevalence of candidiasis, early detection of the causative agents may pave the way for the management of this infection. The present study aimed...
BACKGROUND
Due to the increasing prevalence of candidiasis, early detection of the causative agents may pave the way for the management of this infection. The present study aimed to assess the discriminative power of the six isoenzymatic systems for differentiating the species.
MATERIALS AND METHODS
Sixteen standard and strains and 30 fluconazole-sensitive and fluconazole-resistant clinical strains of were analyzed using a Multilocus Enzyme Electrophoresis (MLEE) method, including six enzymatic systems consisting of malate dehydrogenase (MDH), phosphoglucomutase (PGM), glucose-phosphate isomerase (GPI), glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGD), and malic enzyme (ME).
RESULTS
Among the six enzymatic systems, ME showed no diagnostic activity, whereas MDH provided the best species-specific pattern for species discrimination. In addition, the MDH and G6PD systems provided a discriminatory pattern for differentiating from isolates. The same isoenzymatic activity was detected in all 36 standard and clinical isolates. Moreover, the results showed no correlation between the isoenzymatic profiles and drug resistance.
CONCLUSION
Among the investigated MLEE systems, MDH was able to differentiate between and . Although no association was detected between isoenzyme patterns and fluconazole resistance in this investigation, isoenzyme patterns are likely correlated with virulence factors between species and even within species. To answer these questions, additional studies should be done on more strains.
PubMed: 37434931
DOI: 10.4103/abr.abr_243_22 -
A facile and robust T7-promoter-based high-expression of heterologous proteins in Bacillus subtilis.Bioresources and Bioprocessing May 2022To mimic the Escherichia coli T7 protein expression system, we developed a facile T7 promoter-based protein expression system in an industrial microorganism Bacillus...
To mimic the Escherichia coli T7 protein expression system, we developed a facile T7 promoter-based protein expression system in an industrial microorganism Bacillus subtilis. This system has two parts: a new B. subtilis strain SCK22 and a plasmid pHT7. To construct strain SCK22, the T7 RNA polymerase gene was inserted into the chromosome, and several genes, such as two major protease genes, a spore generation-related gene, and a fermentation foam generation-related gene, were knocked out to facilitate good expression in high-density cell fermentation. The gene of a target protein can be subcloned into plasmid pHT7, where the gene of the target protein was under tight control of the T7 promoter with a ribosome binding site (RBS) sequence of B. subtilis (i.e., AAGGAGG). A few recombinant proteins (i.e., green fluorescent protein, α-glucan phosphorylase, inositol monophosphatase, phosphoglucomutase, and 4-α-glucanotransferase) were expressed with approximately 25-40% expression levels relative to the cellular total proteins estimated by SDS-PAGE by using B. subtilis SCK22/pHT7-derived plasmid. A fed-batch high-cell density fermentation was conducted in a 5-L fermenter, producing up to 4.78 g/L inositol monophosphatase. This expression system has a few advantageous features, such as, wide applicability for recombinant proteins, high protein expression level, easy genetic operation, high transformation efficiency, good genetic stability, and suitability for high-cell density fermentation.
PubMed: 38647747
DOI: 10.1186/s40643-022-00540-4 -
The FEBS Journal Oct 2022Regulation of glycogen metabolism is of vital importance in organisms of all three kingdoms of life. Although the pathways involved in glycogen synthesis and degradation...
Regulation of glycogen metabolism is of vital importance in organisms of all three kingdoms of life. Although the pathways involved in glycogen synthesis and degradation are well known, many regulatory aspects around the metabolism of this polysaccharide remain undeciphered. Here, we used the unicellular cyanobacterium Synechocystis as a model to investigate how glycogen metabolism is regulated in nitrogen-starved dormant cells, which entirely rely on glycogen catabolism to resume growth upon nitrogen repletion. We identified phosphoglucomutase 1 (PGM1) as a key regulatory point in glycogen metabolism, and post-translational modification as an essential mechanism for controlling its activity. We could show that PGM1 is phosphorylated ata residue in the regulatory latch domain (Ser 47) during nitrogen starvation, which inhibits its activity. Inactivation of PGM1 by phosphorylation at Ser 47 prevents premature degradation of the glycogen stores and appears to be essential for survival of Synechocystis in the dormant state. Remarkably, this regulatory mechanism seems to be evolutionary conserved in PGM1 enzymes, from bacteria to humans.
Topics: Glycogen; Humans; Nitrogen; Phosphoglucomutase; Phosphorylation; Synechocystis
PubMed: 35509259
DOI: 10.1111/febs.16471 -
Acta Crystallographica. Section F,... May 2022Phosphoglucomutase 1 (PGM1) plays a central role in glucose homeostasis in human cells. Missense variants of this enzyme cause an inborn error of metabolism, which is...
Phosphoglucomutase 1 (PGM1) plays a central role in glucose homeostasis in human cells. Missense variants of this enzyme cause an inborn error of metabolism, which is categorized as a congenital disorder of glycosylation. Here, two disease-related variants of PGM1, T337M and G391V, which are both located in domain 3 of the four-domain protein, were characterized via X-ray crystallography and biochemical assays. The studies show multiple impacts resulting from these dysfunctional variants, including both short- and long-range structural perturbations. In the T337M variant these are limited to a small shift in an active-site loop, consistent with reduced enzyme activity. In contrast, the G391V variant produces a cascade of structural perturbations, including displacement of both the catalytic phosphoserine and metal-binding loops. This work reinforces several themes that were found in prior studies of dysfunctional PGM1 variants, including increased structural flexibility and the outsized impacts of mutations affecting interdomain interfaces. The molecular mechanisms of PGM1 variants have implications for newly described inherited disorders of related enzymes.
Topics: Catalytic Domain; Crystallography, X-Ray; Glycogen Storage Disease; Humans; Mutation, Missense; Phosphoglucomutase
PubMed: 35506765
DOI: 10.1107/S2053230X22004174 -
The Journal of Biological Chemistry Jun 2022Aspergillus fumigatus is the causative agent of invasive aspergillosis, an infection with mortality rates of up to 50%. The glucan-rich cell wall of A. fumigatus is a...
Aspergillus fumigatus is the causative agent of invasive aspergillosis, an infection with mortality rates of up to 50%. The glucan-rich cell wall of A. fumigatus is a protective structure that is absent from human cells and is a potential target for antifungal treatments. Glucan is synthesized from the donor uridine diphosphate glucose, with the conversion of glucose-6-phosphate to glucose-1-phosphate by the enzyme phosphoglucomutase (PGM) representing a key step in its biosynthesis. Here, we explore the possibility of selectively targeting A. fumigatus PGM (AfPGM) as an antifungal treatment strategy. Using a promoter replacement strategy, we constructed a conditional pgm mutant and revealed that pgm is required for A. fumigatus growth and cell wall integrity. In addition, using a fragment screen, we identified the thiol-reactive compound isothiazolone fragment of PGM as targeting a cysteine residue not conserved in the human ortholog. Furthermore, through scaffold exploration, we synthesized a para-aryl derivative (ISFP10) and demonstrated that it inhibits AfPGM with an IC of 2 μM and exhibits 50-fold selectivity over the human enzyme. Taken together, our data provide genetic validation of PGM as a therapeutic target and suggest new avenues for inhibiting AfPGM using covalent inhibitors that could serve as tools for chemical validation.
Topics: Antifungal Agents; Aspergillosis; Aspergillus fumigatus; Glucans; Humans; Phosphoglucomutase
PubMed: 35504355
DOI: 10.1016/j.jbc.2022.102003