In Silico Drug Screening Analysis against the Overexpression of PGAM1 Gene in Different Cancer Treatments.

Phosphoglycerate mutase 1 (PGAM1) is considered as a novel target for multiple types of cancer drugs for the upregulation in tumor, cell prefoliation, and cell migration. During aerobic glycolysis, PGAM1 plays a critical role in cancer cell metabolism by catalyzing the conversion of 3-phosphoglycerate (3PG) to 2-phosphoglycerate (2PG). In this computational-based study, the molecular docking approach was used with the best binding active sites of PGAM1 to screen 5,000 Chinese medicinal phytochemical library. The docking results were three ligands with docking score, RMSD-refine, and residues. Docking scores were -16.57, -15.22, and -15.74. RMSD values were 0.87, 2.40, and 0.98, and binding site residues were Arg 191, Arg 191, Arg 116, Arg 90, Arg 10, and Tyr 92. The best compounds were subjected to ADMETsar, ProTox-2 server, and Molinspiration analysis to evaluate the toxicological and drug likeliness potential of such selected compounds. The UCSF-Chimera tool was used to visualize the results, which shows that the three medicinal compounds named N-Nitrosohexamethyleneimine, Subtrifloralactone-K, and Kanzonol-N in chain-A were successfully binding with the active pockets of PGAM1. The study might facilitate identifying the hit molecules that could be beneficial in the development of antidrugs against various types of cancer treatment. These hit phytochemicals could be beneficial for further investigation of a novel target for cancer.

Structural analysis of EhPSP in complex with 3-phosphoglyceric acid from Entamoeba histolytica reveals a basis for its lack of phosphoglycerate mutase activity.

Entamoeba histolytica phosphoserine phosphatase (EhPSP), a regulatory enzyme in the serine biosynthetic pathway, is also a structural homolog of cofactor-dependent phosphoglycerate mutase (dPGM). However, despite sharing many of its catalytic residues with dPGM, EhPSP displays no significant mutase activity. In the current work, we determined a crystal structure of EhPSP in complex with 3-PGA to 2.5 Å resolution and observed striking differences between the orientation of 3-PGA bound to EhPSP and that to its other homologous structures. We also performed computational modeling and simulations of the intermediate 2,3-bisphosphoglyceric acid into the active site of EhPSP to better understand its mechanistic details. Based on these results and those of a similar study with the dPGMs from E. coli and B. pseudomallei, the affinity of EhPSP for 2,3-BPG was concluded to be lower than those of the other proteins. Moreover, a different set of 2,3-BPG interacting residues was observed in EhPSP compared to dPGMs, with all of the crucial interacting residues of dPGMs either missing or substituted with weakly interacting residues. This study has expanded our understanding, at the structural level, of the inability of EhPSP to catalyze the mutase reaction and has strengthened earlier conclusions indicating it to be a true phosphatase.

Application of Glyceric Acid to Bio-related Functional Materials and Improvement of Microbial Production.

Glyceric acid (GA) is an oxidative product of glycerol, and its d-isomer is obtained as a phytochemical from tobacco leaves and fruits of some plants. However, the production and applications of GA have not yet been fully investigated. In this review, recent developments in the microbial production of GA and its application to bio-related materials are summarized. The sodium salt of diacylated GA showed superior surface tension-lowering activity and antitrypsin activity. GA and its glucosyl derivative had positive effects on the viability and collagen production of skin cells in vitro, respectively. Glucosyl derivatives of GA showed protective effects against heat-induced protein aggregation. In addition, the microbial production of GA using raw glycerol as the starting material was investigated. The effect of methanol, a major impurity in raw glycerol, on GA production was investigated, and mutant strains to tolerate methanol in the culture were constructed. Enantioselective production of GA using newly isolated microbial strains has also been developed.

Molecular simulations unravel the molecular principles that mediate selective permeability of carboxysome shell protein.

Bacterial microcompartments (BMCs) are nanoscale proteinaceous organelles that encapsulate enzymes from the cytoplasm using an icosahedral protein shell that resembles viral capsids. Of particular interest are the carboxysomes (CBs), which sequester the CO2-fixing enzymes ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) to enhance carbon assimilation. The carboxysome shell serves as a semi-permeable barrier for passage of metabolites in and out of the carboxysome to enhance CO2 fixation. How the protein shell directs influx and efflux of molecules in an effective manner has remained elusive. Here we use molecular dynamics and umbrella sampling calculations to determine the free-energy profiles of the metabolic substrates, bicarbonate, CO2 and ribulose bisphosphate and the product 3-phosphoglycerate associated with their transition through the major carboxysome shell protein CcmK2. We elucidate the electrostatic charge-based permeability and key amino acid residues of CcmK2 functioning in mediating molecular transit through the central pore. Conformational changes of the loops forming the central pore may also be required for transit of specific metabolites. The importance of these in-silico findings is validated experimentally by site-directed mutagenesis of the key CcmK2 residue Serine 39. This study provides insight into the mechanism that mediates molecular transport through the shells of carboxysomes, applicable to other BMCs. It also offers a predictive approach to investigate and manipulate the shell permeability, with the intent of engineering BMC-based metabolic modules for new functions in synthetic biology.

Mapping the intracellular metabolome of yeast biocapsules - Spherical structures of yeast attached to fungal pellets.

Co-culture conditions are beneficial for study due to the advances which arise from symbiotic interactions and which cannot be replicated under pure culture conditions. Here, the focus is on the connection between two fungi - a yeast, Saccharomyces cerevisiae, and a filamentous fungus, Penicillium chrysogenum - in a yeast immobilization system termed' yeast biocapsules', where the yeast and filamentous fungus are strongly attached to one another, forming spherical structures. This co-culture condition hinders filamentous fungal biomass growth, while immobilization of yeast cells continues to increase. The effect of the co-culture condition on endometabolites or intracellular metabolites were tracked during the beginning and end of the yeast biocapsule formation period, and metabolites analyzed by Gas Chromatography-Mass Spectrometry Detector (GC-MSD). Distinct metabolite profiles were found between single culture conditions, involving each organism separately, and with the co-culture condition, where there were differences in 54 endometabolites. Specifically, co-culture condition compounds such as fructose, glycolic acid and glyceric acid were present in higher concentrations at the end of biocapsule formation. These results shed light on the mechanisms and biochemical impact of the interaction between the yeast and filamentous fungus and serve as a basis to apply and further develop yeast biocapsules as a new biotechnological tool with benefits for industry.

Perturbation of phosphoglycerate kinase 1 (PGK1) only marginally affects glycolysis in cancer cells.

Phosphoglycerate kinase 1 (PGK1) plays important roles in glycolysis, yet its forward reaction kinetics are unknown, and its role especially in regulating cancer cell glycolysis is unclear. Here, we developed an enzyme assay to measure the kinetic parameters of the PGK1-catalyzed forward reaction. The Km values for 1,3-bisphosphoglyceric acid (1,3-BPG, the forward reaction substrate) were 4.36 µm (yeast PGK1) and 6.86 µm (human PKG1). The Km values for 3-phosphoglycerate (3-PG, the reverse reaction substrate and a serine precursor) were 146 µm (yeast PGK1) and 186 µm (human PGK1). The Vmax of the forward reaction was about 3.5- and 5.8-fold higher than that of the reverse reaction for the human and yeast enzymes, respectively. Consistently, the intracellular steady-state concentrations of 3-PG were between 180 and 550 µm in cancer cells, providing a basis for glycolysis to shuttle 3-PG to the serine synthesis pathway. Using siRNA-mediated PGK1-specific knockdown in five cancer cell lines derived from different tissues, along with titration of PGK1 in a cell-free glycolysis system, we found that the perturbation of PGK1 had no effect or only marginal effects on the glucose consumption and lactate generation. The PGK1 knockdown increased the concentrations of fructose 1,6-bisphosphate, dihydroxyacetone phosphate, glyceraldehyde 3-phosphate, and 1,3-BPG in nearly equal proportions, controlled by the kinetic and thermodynamic states of glycolysis. We conclude that perturbation of PGK1 in cancer cells insignificantly affects the conversion of glucose to lactate in glycolysis.

Metabolic Pathway Rerouting in Paraburkholderia rhizoxinica Evolved Long-Overlooked Derivatives of Coenzyme F420.

Coenzyme F420 is a specialized redox cofactor with a negative redox potential. It supports biochemical processes like methanogenesis, degradation of xenobiotics, and the biosynthesis of antibiotics. Although well-studied in methanogenic archaea and actinobacteria, not much is known about F420 in Gram-negative bacteria. Genome sequencing revealed F420 biosynthetic genes in the Gram-negative, endofungal bacterium Paraburkholderia rhizoxinica, a symbiont of phytopathogenic fungi. Fluorescence microscopy, high-resolution LC-MS, and structure elucidation by NMR demonstrated that the encoded pathway is active and yields unexpected derivatives of F420 (3PG-F420). Further analyses of a biogas-producing microbial community showed that these derivatives are more widespread in nature. Genetic and biochemical studies of their biosynthesis established that a specificity switch in the guanylyltransferase CofC reprogrammed the pathway to start from 3-phospho-d-glycerate, suggesting a rerouting event during the evolution of F420 biosynthesis. Furthermore, the cofactor activity of 3PG-F420 was validated, thus opening up perspectives for its use in biocatalysis. The 3PG-F420 biosynthetic gene cluster is fully functional in Escherichia coli, enabling convenient production of the cofactor by fermentation.

Introducing an Experimental Design Approach for Efficient Optimization of Chiral Derivatization Conditions for D- and L-Glyceric Acids.

A sensitive analytical method was developed for individual analyses of D- and L-glyceric acids by chiral derivatization - liquid chromatography-tandem mass spectrometry. To elucidate rapid and efficient optimization for derivatization we newly introduced a concept of design of experiments (DOE). The optimization of major 5 factors in the derivatization could be predicted with only 28 measurements. By applying DOE to optimization, the yields of desired derivatives increased five-fold against before optimization.

Synthesis and Characterization of a Novel Glycolipid with Glucosylglycerate as a Hydrophile Showing Protective Effects on Heat-induced Protein Denaturation.

A novel glycolipid featuring a glucosylglycerate moiety as a polar head group was synthesized in two steps from sucrose, glycerate, and N-dodecylamine. Glucosylglyceric acid was formed from sucrose and glyceric acid using sucrose synthase as a catalyst, followed by condensation with N-dodecylamine using 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) as a condensing agent. A white solid compound was recovered with a yield of 21% after purification by hydrophobic column chromatography. The structure and purity of the isolated compound, identified as N-dodecyl glucosylglyceric acid amide (aGGA), were confirmed by 1H and 13C nuclear magnetic resonance and liquid chromatography-electrospray ionization-mass spectrometry. aGGA was soluble in several polar solvents, including acetone, dimethyl formamide, and short chain alcohols. The dissolution of aGGA in water reduced the surface tension to 27.8 mN m-1 at a critical micellar concentration of 1.57 × 10-4 M. In addition, the presence of aGGA at concentrations as low at 0.68 mM protected egg white from heat-induced denaturation. These results suggest that aGGA could be useful as a protein-protecting surfactant.

Production of mannosylglycerate in Saccharomyces cerevisiae by metabolic engineering and bioprocess optimization.

BACKGROUND: Mannosylglycerate (MG) is one of the most widespread compatible solutes among marine microorganisms adapted to hot environments. This ionic solute holds excellent ability to protect proteins against thermal denaturation, hence a large number of biotechnological and clinical applications have been put forward. However, the current prohibitive production costs impose severe constraints towards large-scale applications. All known microbial producers synthesize MG from GDP-mannose and 3-phosphoglycerate via a two-step pathway in which mannosyl-3-phosphoglycerate is the intermediate metabolite. In an early work, this pathway was expressed in Saccharomyces cerevisiae with the goal to confirm gene function (Empadinhas et al. in J Bacteriol 186:4075-4084, 2004), but the level of MG accumulation was low. Therefore, in view of the potential biotechnological value of this compound, we decided to invest further effort to convert S. cerevisiae into an efficient cell factory for MG production. RESULTS: To drive MG production, the pathway for the synthesis of GDP-mannose, one of the MG biosynthetic precursors, was overexpressed in S. cerevisiae along with the MG biosynthetic pathway. MG production was evaluated under different cultivation modes, i.e., flask bottle, batch, and continuous mode with different dilution rates. The genes encoding mannose-6-phosphate isomerase (PMI40) and GDP-mannose pyrophosphorylase (PSA1) were introduced into strain MG01, hosting a plasmid encoding the MG biosynthetic machinery. The resulting engineered strain (MG02) showed around a twofold increase in the activity of PMI40 and PSA1 in comparison to the wild-type. In batch mode, strain MG02 accumulated 15.86 mgMG g DCW -1 , representing a 2.2-fold increase relative to the reference strain (MG01). In continuous culture, at a dilution rate of 0.15 h-1, there was a 1.5-fold improvement in productivity. CONCLUSION: In the present study, the yield and productivity of MG were increased by overexpression of the GDP-mannose pathway and optimization of the mode of cultivation. A maximum of 15.86 mgMG g DCW -1 was achieved in batch cultivation and maximal productivity of 1.79 mgMG g DCW -1  h-1 in continuous mode. Additionally, a positive correlation between MG productivity and growth rate/dilution rate was established, although this correlation is not observed for MG yield.

Characterization of an Unusual Glycerate Esterification Process in Vioprolide Biosynthesis.

Bacteria produce a large number of secondary metabolites with extraordinary chemical structures and bioactivities. Vioprolides are promising anticancer and antifungal lead compounds produced by the myxobacterium Cystobacter violaceus Cb vi35, which are initially synthesized as acylated precursors (previoprolides) by nonribosomal peptide synthetases (NRPS). Here, we describe and characterize an unprecedented glycerate esterification process in the biosynthesis of vioprolides. In vitro biochemical investigations revealed that the fatty acyl chain of previoprolides is adenylated by the starting fatty acyl-AMP ligase (FAAL) domain, while the glycerate moiety is incorporated by the FkbH domain. An unusual ester-bond forming condensation domain is shown responsible for the acylation of glycerate. LC-MS analysis and bioactivity assays suggest that the acylation serves for directed membrane transport rather than representing a prodrug mechanism.

The phosphoglycerate kinase 1 variants found in carcinoma cells display different catalytic activity and conformational stability compared to the native enzyme.

Cancer cells are able to survive in difficult conditions, reprogramming their metabolism according to their requirements. Under hypoxic conditions they shift from oxidative phosphorylation to aerobic glycolysis, a behavior known as Warburg effect. In the last years, glycolytic enzymes have been identified as potential targets for alternative anticancer therapies. Recently, phosphoglycerate kinase 1 (PGK1), an ubiquitous enzyme expressed in all somatic cells that catalyzes the seventh step of glycolysis which consists of the reversible phosphotransfer reaction from 1,3-bisphosphoglycerate to ADP, has been discovered to be overexpressed in many cancer types. Moreover, several somatic variants of PGK1 have been identified in tumors. In this study we analyzed the effect of the single nucleotide variants found in cancer tissues on the PGK1 structure and function. Our results clearly show that the variants display a decreased catalytic efficiency and/or thermodynamic stability and an altered local tertiary structure, as shown by the solved X-ray structures. The changes in the catalytic properties and in the stability of the PGK1 variants, mainly due to the local changes evidenced by the X-ray structures, suggest also changes in the functional role of PGK to support the biosynthetic need of the growing and proliferating tumour cells.

Structure of the triose-phosphate/phosphate translocator reveals the basis of substrate specificity.

The triose-phosphate/phosphate translocator (TPT) catalyses the strict 1:1 exchange of triose-phosphate, 3-phosphoglycerate and inorganic phosphate across the chloroplast envelope, and plays crucial roles in photosynthesis. Despite rigorous study for more than 40 years, the molecular mechanism of TPT is poorly understood because of the lack of structural information. Here we report crystal structures of TPT bound to two different substrates, 3-phosphoglycerate and inorganic phosphate, in occluded conformations. The structures reveal that TPT adopts a 10-transmembrane drug/metabolite transporter fold. Both substrates are bound within the same central pocket, where conserved lysine, arginine and tyrosine residues recognize the shared phosphate group. A structural comparison with the outward-open conformation of the bacterial drug/metabolite transporter suggests a rocker-switch motion of helix bundles, and molecular dynamics simulations support a model in which this rocker-switch motion is tightly coupled to the substrate binding, to ensure strict 1:1 exchange. These results reveal the unique mechanism of sugar phosphate/phosphate exchange by TPT.

FORMULATION AND TECHNOLOGY DEVELOPMENT OF HERBAL PHENOLIC BIOPOLYMER-CONTAINING FILMS FOR BURN TREATMENT.

Application of phytofilms based on biosolublepolymers is considered as a prospectivemethod for burn treatment . Herbal remedies contain biologically active substances, that are relatively less toxic, do not cause skin irritation or allergic reactions and, importantly, affectstrains of the microorganisms and viruses resistant to antibiotics and synthetic drugs. Nowadays, the advantages are given to such burn healing drugs, which along with high specific efficacy, have analgesic, anti-inflammatory and antimicrobial effects, and don't irritate the tissues. The mentioned peculiarities are characteristic for a new herbal phenolic biopolymer poly[3-(3,4-dihydroxyphenyl) glyceric acid](PDGA), isolated from the roots and stems of different comfrey species . The aim of the study was the development of the formulation and technology of biosoluble films for burn treatment on the basis of PDGA. The optimal content of phytofilm for burn healing was selected on the basis of the biopharmaceutical study results. The impact of the film-former on the quality, adhesion and moisture absorption of the phytofilmhas been studied. The optimal degree of the phytofilm moisture, determining its high adhesive properties,was established. The film prepared on the basis of sodium alginate, with 30.4% humidity, demonstrated the greatest adhesion strength. After investigation of the PDGA release it was found, that the hydrophilic bases such as: sodium carboxymethyl-cellulose (69.2%) andsodium alginate (78,65%) appeared to be optimal among the others. At the same time, taking into consideration the disadvantages of sodium carboxymethyl-cellulose (tautening effect on burnt surface, relatively low stability), a film based on sodium alginate has been chosen. The manufacturing technology for obtaining PDGA-containing phytofilm by casting is proposed. Theshelf-lifeofproposedPDGA-containingphytofilmis 2 years.

Hyperpolarized Sodium [1-13C]-Glycerate as a Probe for Assessing Glycolysis In Vivo.

Hyperpolarized 13C magnetic resonance spectroscopy (MRS) provides unprecedented opportunities to obtain clinical diagnostic information through in vivo monitoring of metabolic pathways. The continuing advancement of this field relies on the identification of molecular probes that can effectively interrogate pathways critical to disease. In this report, we describe the synthesis, development, and in vivo application of sodium [1-13C]-glycerate ([13C]-Glyc) as a novel probe for evaluating glycolysis using hyperpolarized 13C MRS. This agent was prepared by a concise synthetic route and formulated for dynamic nuclear polarization. [13C]-Glyc displayed a high level of polarization and long spin-lattice relaxation time-both of which are necessary for future clinical investigations. In vivo spectroscopic studies with hyperpolarized [13C]-Glyc in rat liver furnished metabolic products, [13C]-labeled pyruvate and lactate, originating from glycolysis. The levels of production and relative intensities of these metabolites were directly correlated with the induced glycolytic state (fasted versus fed groups). This work establishes hyperpolarized [13C]-Glyc as a novel agent for clinically relevant 13C MRS studies of energy metabolism and further provides opportunities for evaluating intracellular redox states in biochemical investigations.

Crystal structure of enolase from Drosophila melanogaster.

Enolase is an important enzyme in glycolysis and various biological processes. Its dysfunction is closely associated with diseases. Here, the enolase from Drosophila melanogaster (DmENO) was purified and crystallized. A crystal of DmENO diffracted to 2.0 Šresolution and belonged to space group R32. The structure was solved by molecular replacement. Like most enolases, DmENO forms a homodimer with conserved residues in the dimer interface. DmENO possesses an open conformation in this structure and contains conserved elements for catalytic activity. This work provides a structural basis for further functional and evolutionary studies of enolase.

Biocatalytic Asymmetric Phosphorylation Catalyzed by Recombinant Glycerate-2-Kinase.

The efficient synthesis of pure d-glycerate-2-phosphate is of great interest due to its importance as an enzyme substrate and metabolite. Therefore, we investigated a straightforward one-step biocatalytic phosphorylation of glyceric acid. Glycerate-2-kinase from Thermotoga maritima was expressed in Escherichia coli, allowing easy purification. The selective glycerate-2-kinase-catalyzed phosphorylation was followed by 31 P NMR and showed excellent enantioselectivity towards phosphorylation of the d-enantiomer of glyceric acid. This straightforward phosphorylation reaction and subsequent product isolation enabled the preparation of enantiomerically pure d-glycerate 2-phosphate. This phosphorylation reaction, using recombinant glycerate-2-kinase, yielded d-glycerate 2-phosphate in fewer reaction steps and with higher purity than chemical routes.

DEVELOPMENT OF FORMULATION AND TECHNOLOGY FOR THE POLY[3-(3,4-DIHYDROXYPHENYL)GLYCERIC ACID] GEL.

One of the most actual problems of pharmacy is the development of medication forms for external application with complex effects on (gel, emplastro, aerosol, etc.) skin wounds, burns and inflammatory factors. The centuries-old practice of using phyto-preparations (herbal remedies) proved that they have fewer side effects in comparison with synthetic drugs. Despite the wide application of herbal preparations, in the literature there is a little information about their application in development of wound and burn healing modern dosage forms. Among the medicinal plants with the mentioned pharmacological actions, comfrey (Symphytum L.) should be distinguished. Phenolic polymer poly[3-(3,4-dihydroxyphenyl)glyceric acid] (PDGA) or poly[oxy-1-carboxy-2-(3,4-dihydroxyphenyl)ethylene], amounting approximately 25% of polysaccharides and 1.5-2.5% of dry plant material, were isolated from the roots and stems of Caucasian comfrey species (S. asperum, S. caucasicum). Contrary to polysaccharides this phenolic polymer of Comfrey appeared to have a high immunomodulatory (anticomplement), antioxidative, antilipoperoxidantive, anti-inflammatory and wound-healing efficacy/activities. The aim of the study was development of the composition and technology of PDGA-containing gel. According to the results of complex biopharmaceutical studies PDGA gel optimal composition has been proved. The technological scheme for preparation of PDGA gel has been developed. PDGA gel stability under normal conditions of storage at +40С was studied. The gel has a shelf life (determined expiration date) of 2 year.

Molecular dynamics simulation reveals how phosphorylation of tyrosine 26 of phosphoglycerate mutase 1 upregulates glycolysis and promotes tumor growth.

Phosphoglycerate mutase 1 (PGAM1) catalyzes the eighth step of glycolysis and is often found upregulated in cancer cells. To test the hypothesis that the phosphorylation of tyrosine 26 residue of PGAM1 greatly enhances its activity, we performed both conventional and steered molecular dynamics simulations on the binding and unbinding of PGAM1 to its substrates, with tyrosine 26 either phosphorylated or not. We analyzed the simulated data in terms of structural stability, hydrogen bond formation, binding free energy, etc. We found that tyrosine 26 phosphorylation enhances the binding of PGAM1 to its substrates through generating electrostatic environment and structural features that are advantageous to the binding. Our results may provide valuable insights into computer-aided design of drugs that specifically target cancer cells with PGAM1 tyrosine 26 phosphorylated.

Synthesis and biological evaluation of enantiomerically pure glyceric acid derivatives as LpxC inhibitors.

Inhibitors of the UDP-3-O-[(R)-3-hydroxymyristoyl]-N-acetylglucosamine deacetylase (LpxC) represent a promising class of novel antibiotics, selectively combating Gram-negative bacteria. In order to elucidate the impact of the hydroxymethyl groups of diol (S,S)-4 on the inhibitory activity against LpxC, glyceric acid ethers (R)-7a, (S)-7a, (R)-7b, and (S)-7b, lacking the hydroxymethyl group in benzylic position, were synthesized. The compounds were obtained in enantiomerically pure form by a chiral pool synthesis and a lipase-catalyzed enantioselective desymmetrization, respectively. The enantiomeric hydroxamic acids (R)-7b (Ki=230nM) and (S)-7b (Ki=390nM) show promising enzyme inhibition. However, their inhibitory activities do not substantially differ from each other leading to a low eudismic ratio. Generally, the synthesized glyceric acid derivatives 7 show antibacterial activities against two Escherichia coli strains exceeding the ones of their respective regioisomes 6.