Biochemical and cellular studies of three human 3-phosphoglycerate dehydrogenase variants responsible for pathological reduced L-serine levels.

In the brain, the non-essential amino acid L-serine is produced through the phosphorylated pathway (PP) starting from the glycolytic intermediate 3-phosphoglycerate: among the different roles played by this amino acid, it can be converted into D-serine and glycine, the two main co-agonists of NMDA receptors. In humans, the enzymes of the PP, namely phosphoglycerate dehydrogenase (hPHGDH, which catalyzes the first and rate-limiting step of this pathway), 3-phosphoserine aminotransferase, and 3-phosphoserine phosphatase are likely organized in the cytosol as a metabolic assembly (a "serinosome"). The hPHGDH deficiency is a pathological condition biochemically characterized by reduced levels of L-serine in plasma and cerebrospinal fluid and clinically identified by severe neurological impairment. Here, three single-point variants responsible for hPHGDH deficiency and Neu-Laxova syndrome have been studied. Their biochemical characterization shows that V261M, V425M, and V490M substitutions alter either the kinetic (both maximal activity and Km for 3-phosphoglycerate in the physiological direction) and the structural properties (secondary, tertiary, and quaternary structure, favoring aggregation) of hPHGDH. All the three variants have been successfully ectopically expressed in U251 cells, thus the pathological effect is not due to hindered expression level. At the cellular level, mistargeting and aggregation phenomena have been observed in cells transiently expressing the pathological protein variants, as well as a reduced L-serine cellular level. Previous studies demonstrated that the pharmacological supplementation of L-serine in hPHGDH deficiencies could ameliorate some of the related symptoms: our results now suggest the use of additional and alternative therapeutic approaches.

A serum metabolic biomarker panel for early rheumatoid arthritis.

Objective: There is an urgent need for novel biomarkers to improve the early diagnosis of rheumatoid arthritis (ERA). Current serum biomarkers used in the management of ERA, including rheumatoid factor and anti-cyclic citrullinated peptide (ACPA), show limited specificity and sensitivity. Here, we used metabolomics to uncover new serum biomarkers of ERA. Methods: We applied an untargeted metabolomics approach including gas chromatography time-of-flight mass spectrometry in serum samples from an ERA cohort (n=32) and healthy controls (n=19). Metabolite set enrichment analysis was performed to explore potentially important biological pathways. Partial least squares discriminant analysis and variable importance in projection analysis were performed to construct an ERA biomarker panel. Results: Significant differences in the content of 11/81 serum metabolites were identified in patients with ERA. Receiver operating characteristic (ROC) analysis showed that a panel of only three metabolites (glyceric acid, lactic acid, and 3-hydroxisovaleric acid) could correctly classify 96.7% of patients with ERA, with an area under the ROC curve of 0.963 and with 94.4% specificity and 93.5% sensitivity, outperforming ACPA-based diagnosis by 2.9% and, thus, improving the preclinical detection of ERA. Aminoacyl-tRNA biosynthesis and serine, glycine, and phenylalanine metabolism were the most significant dysregulated pathways in patients with ERA. Conclusion: A metabolomics serum-based biomarker panel composed of glyceric acid, lactic acid, and 3-hydroxisovaleric acid offers potential for the early clinical diagnosis of RA.

Controlling the confounding effect of metabolic gene expression to identify actual metabolite targets in microsatellite instability cancers.

BACKGROUND: The metabolome is the best representation of cancer phenotypes. Gene expression can be considered a confounding covariate affecting metabolite levels. Data integration across metabolomics and genomics to establish the biological relevance of cancer metabolism is challenging. This study aimed to eliminate the confounding effect of metabolic gene expression to reflect actual metabolite levels in microsatellite instability (MSI) cancers. METHODS: In this study, we propose a new strategy using covariate-adjusted tensor classification in high dimensions (CATCH) models to integrate metabolite and metabolic gene expression data to classify MSI and microsatellite stability (MSS) cancers. We used datasets from the Cancer Cell Line Encyclopedia (CCLE) phase II project and treated metabolomic data as tensor predictors and data on gene expression of metabolic enzymes as confounding covariates. RESULTS: The CATCH model performed well, with high accuracy (0.82), sensitivity (0.66), specificity (0.88), precision (0.65), and F1 score (0.65). Seven metabolite features adjusted for metabolic gene expression, namely, 3-phosphoglycerate, 6-phosphogluconate, cholesterol ester, lysophosphatidylethanolamine (LPE), phosphatidylcholine, reduced glutathione, and sarcosine, were found in MSI cancers. Only one metabolite, Hippurate, was present in MSS cancers. The gene expression of phosphofructokinase 1 (PFKP), which is involved in the glycolytic pathway, was related to 3-phosphoglycerate. ALDH4A1 and GPT2 were associated with sarcosine. LPE was associated with the expression of CHPT1, which is involved in lipid metabolism. The glycolysis, nucleotide, glutamate, and lipid metabolic pathways were enriched in MSI cancers. CONCLUSIONS: We propose an effective CATCH model for predicting MSI cancer status. By controlling the confounding effect of metabolic gene expression, we identified cancer metabolic biomarkers and therapeutic targets. In addition, we provided the possible biology and genetics of MSI cancer metabolism.

Pt1 enhanced C-H activation synergistic with Ptn catalysis for glycerol cascade oxidation to glyceric acid.

The selective oxidation of glycerol to glyceric acid, an important value-added reaction from polyols, is a typical cascade catalytic process. It is still of great challenge to simultaneously achieve high glycerol activity and glyceric acid selectivity, suffering from either deep oxidation and C-C cleavage or poor oxidation efficiency from glyceraldehyde to glyceric acid. Herein, this work, inspired by nature, proposes a cascade synergistic catalysis strategy by atomic and low-coordinated cluster Pt on well-defined Cu-CuZrOx, which involves enhanced C-H activation on atomic Pt1 and O-H activation on cluster Ptn in the oxidation of glycerol to glyceraldehyde, and cluster Ptn for C=O activation followed by O-H insertion and atomic Pt1 for C-H activation in the tandem oxidation of glyceraldehyde to glyceric acid. The enhanced C-H activation in the cascade process by atomic Pt1 is revealed to be essential for the high glycerol activity (90.0±0.1%) and the glyceric acid selectivity (80.2±0.2%).

Tissue-specific transcriptional analysis outlines calcium-induced core metabolic changes in sweet cherry fruit.

The role of calcium in fruit ripening has been established, however knowledge regarding the molecular analysis at fruit tissue-level is still lacking. To address this, we examined the impact of foliar-applied calcium (0.5% CaCl2) in the ripening metabolism in skin and flesh tissues of the sweet cherry 'Tragana Edessis' fruit at the harvest stage. Exogenously applied calcium increased endogenous calcium level in flesh tissue and reduced fruit respiration rate and cracking traits. Fruit metabolomic along with transcriptomic analysis unraveled common and tissue-specific metabolic pathways associated with calcium feeding. Treatment with calcium diminished several alcohols (arabitol, sorbitol), sugars (fructose, maltose), acids (glyceric acid, threonic acid) and increased ribose and proline in both fruit tissues. Moreover, numerous primary metabolites, such as proline and galacturonic acid, were differentially accumulated in calcium-exposed tissues. Calcium-affected genes that involved in ubiquitin/ubl conjugation and cell wall biogenesis/degradation were differentially expressed between skin and flesh samples. Notably, skin and flesh tissues shared common calcium-responsive genes and exhibited substantial similarity in their expression patterns. In both tissues, calcium activated gene expression, most strongly those involved in plant-pathogen interaction, plant hormone signaling and MAPK signaling pathway, thus affecting related metabolic processes. By contrast, calcium depressed the expression of genes related to TCA cycle, oxidative phosphorylation, and starch/sucrose metabolism in both tissues. This work established both calcium-driven common and specialized metabolic suites in skin and flesh cherry tissues, demonstrating the utility of this approach to characterize fundamental aspects of calcium in fruit physiology.

Caffeic and 3-(3,4-dihydroxyphenyl)glyceric acid derivatives as antimicrobial agent: biological evaluation and molecular docking studies.

Herein we report the evaluation of the antimicrobial activity of some previously synthesized 3-(3,4-dihydroxyphenyl)glyceric acid in benzylated and in free 3,4 hydroxy groups in catechol moiety along with some caffeic and 3-(3,4-dihydroxyphenyl)glyceric acid amides using the microdilution method. The evaluation revealed that compounds showed in general moderate to low activity with MIC in range of 0.36-4.5 mg/mL. Compounds were also studied against three resistant bacteria strains MRSA (Methicillin-resistant Staphylococcus aureus), E. coli and P. aeruginosa. Seven out of ten compounds were more potent than reference drugs ampicillin and streptomycin against MRSA, while against another two resistant strains seven compounds showed low activity and the rest were inactive. Antifungal activity of the tested compounds was much better than antibacterial, with MIC in the range of 0.019-3.0 mg/mL. Compounds #7 and 15 showed good activity against all fungi tested, being more potent than ketoconazole and in some case even better than bifonazole used as reference drugs. Docking studies revealed that the most active compound #7 binds to the haem group of the enzyme in the same way as ketoconazole.

Investigation of salivary biomarkers as indicators of skeletal and dental maturity in children.

OBJECTIVE: Estimation of patient's skeletal maturity in orthodontics is essential for the diagnosis and treatment planning. The aim of the study was to investigate the potential use of metabolic fingerprint of saliva for bone growth and tooth development estimation. MATERIALS AND METHODS: Saliva samples from 54 young patients were analysed by an untargeted gas chromatography-mass spectrometry metabolomics-based method. The skeletal maturity was calculated with the cervical vertebrae maturation method, and the dental age was estimated with the Demirjian method. Multivariate analysis and univariate analysis were performed to investigate differences within skeletal, dental and chronological age groups. RESULTS: Metabolomic analysis identified 61 endogenous compounds. Mannose, glucose, glycerol, glyceric acid and pyroglutamic acid levels differentiated significantly with skeletal age (P = .02 to .043), while mannose, lactic acid, glycolic acid, proline, norleucine, 3-aminoisobutyric acid, threonine, cadaverine and hydrocinnamic acid levels differed within the dental age groups (P = .018 to .04); according to the chronological age, only the levels of mannose and 3-hydroxyphenylacetic acid showed variation (P = .029 and .048). The principal component analysis did not manage to highlight differences between the groups of the studied parameters. CONCLUSION: Differentiated levels of mannose, glucose, glycerol, glyceric acid and pyroglutamic acid related to skeletal maturation were identified. According to dental development, the levels of mannose, lactic acid, glycolic acid, proline, norleucine, 3-aminoisobutyric acid, threonine, cadaverine and hydrocinnamic acid differed within the groups, while regarding chronological age, only the levels of mannose and 3-hydroxyphenylacetic acid showed variations. Further studies are required to prove their relation to skeletal and dental development pathway by applying complementary analytical techniques to wider cover the metabolome.

Identification of a Novel Alditol Oxidase from Thermopolyspora flexuosa with Potential Application in D-Glyceric Acid Production.

Glycerol is a potential sustainable feedstock, and biorefining processes to convert glycerol into value-added chemicals have been developed over the past decade. Alditol oxidase (AldO) is capable of selectively oxidizing the primary hydroxyl groups of alditols such as glycerol. In this study, a new FAD-binding protein from Thermopolyspora flexuosa was expressed and identified as a novel alditol oxidase (AldOT. fle). AldOT. fle displayed the optimal activity at pH 8.0 and 25 °C. AldOT. fle was not metal-dependent, but the activity was completely inhibited by Fe3+. AldOT. fle had a wide substrate specificity and high catalytic efficiency for glycerol. Furthermore, the recombinant AldOT. fle could produce D-glyceric acid from glycerol with a conversion rate ranging from 86.6% (5 mM glycerol) to 20.5% (500 mM glycerol). The recombinant E. coli with AldOT. fle could also produce 23.8 mM D-glyceric acid from 100 mM glycerol. The recombinant AldOT. fle had the potential to produce other aldehyde products by selectively oxidizing the hydroxyl groups of alditols and many other commodity chemicals by redesigning glycerol metabolism.

Parkinson's disease protein PARK7 prevents metabolite and protein damage caused by a glycolytic metabolite.

Cells are continuously exposed to potentially dangerous compounds. Progressive accumulation of damage is suspected to contribute to neurodegenerative diseases and aging, but the molecular identity of the damage remains largely unknown. Here we report that PARK7, an enzyme mutated in hereditary Parkinson's disease, prevents damage of proteins and metabolites caused by a metabolite of glycolysis. We found that the glycolytic metabolite 1,3-bisphosphoglycerate (1,3-BPG) spontaneously forms a novel reactive intermediate that avidly reacts with amino groups. PARK7 acts by destroying this intermediate, thereby preventing the formation of proteins and metabolites with glycerate and phosphoglycerate modifications on amino groups. As a consequence, inactivation of PARK7 (or its orthologs) in human cell lines, mouse brain, and Drosophila melanogaster leads to the accumulation of these damaged compounds, most of which have not been described before. Our work demonstrates that PARK7 function represents a highly conserved strategy to prevent damage in cells that metabolize carbohydrates. This represents a fundamental link between metabolism and a type of cellular damage that might contribute to the development of Parkinson's disease.

Concentration and pH effect on the electronic circular dichroism and anisotropy spectra of aqueous solutions of glyceric acid calcium salt.

Electronic circular dichroism (ECD) and anisotropy spectra carry information on differential absorption of left- and right-circularly polarized light (LCPL and RCPL) by optically active compounds. This makes them powerful tools for the rapid determination of enantiomeric excesses (ee) in asymmetric synthetic and pharmaceutical chemistry, as well as for predicting the ee inducible by ultraviolet (UV) CPL. The ECD response of a chiral molecule is, however, critically dependent on the properties of the surrounding medium. Here, we report on the first ECD/anisotropy spectra of aqueous solutions of the calcium salt dihydrate of glyceric acid. A systematic study of the effect of the salt concentration and pH on the chiroptical response revealed significant changes and the appearance of a new ECD band of opposite sign. Based on the literature, this can be rationalized by the increase in the relative proportion of free glyceric acid/glycerate to Ca2+ complexes with glycerate with decreasing salt concentration or pH. Glyceric acid can be readily produced under astrophysical conditions. The anisotropy spectra of the solution containing prevalently the free form of this dihydroxy carboxylic acid resemble the ones of previously investigated aliphatic chain hydroxycarboxylic acids and proteinogenic amino acids. This indicates possible common handedness of stellar CPL-induced asymmetry in the potential comonomers of primitive proto-peptides.

Molecular mechanism of glycolytic flux control intrinsic to human phosphoglycerate kinase.

Glycolysis plays a fundamental role in energy production and metabolic homeostasis. The intracellular [adenosine triphosphate]/[adenosine diphosphate] ([ATP]/[ADP]) ratio controls glycolytic flux; however, the regulatory mechanism underlying reactions catalyzed by individual glycolytic enzymes enabling flux adaptation remains incompletely understood. Phosphoglycerate kinase (PGK) catalyzes the reversible phosphotransfer reaction, which directly produces ATP in a near-equilibrium step of glycolysis. Despite extensive studies on the transcriptional regulation of PGK expression, the mechanism in response to changes in the [ATP]/[ADP] ratio remains obscure. Here, we report a protein-level regulation of human PGK (hPGK) by utilizing the switching ligand-binding cooperativities between adenine nucleotides and 3-phosphoglycerate (3PG). This was revealed by nuclear magnetic resonance (NMR) spectroscopy at physiological salt concentrations. MgADP and 3PG bind to hPGK with negative cooperativity, whereas MgAMPPNP (a nonhydrolyzable ATP analog) and 3PG bind to hPGK with positive cooperativity. These opposite cooperativities enable a shift between different ligand-bound states depending on the intracellular [ATP]/[ADP] ratio. Based on these findings, we present an atomic-scale description of the reaction scheme for hPGK under physiological conditions. Our results indicate that hPGK intrinsically modulates its function via ligand-binding cooperativities that are finely tuned to respond to changes in the [ATP]/[ADP] ratio. The alteration of ligand-binding cooperativities could be one of the self-regulatory mechanisms for enzymes in bidirectional pathways, which enables rapid adaptation to changes in the intracellular environment.

Biophysical and biochemical properties of PHGDH revealed by studies on PHGDH inhibitors.

The rate-limiting serine biogenesis enzyme PHGDH is overexpressed in cancers. Both serine withdrawal and genetic/pharmacological inhibition of PHGDH have demonstrated promising tumor-suppressing activities. However, the enzyme properties of PHGDH are not well understood and the discovery of PHGDH inhibitors is still in its infancy. Here, oridonin was identified from a natural product library as a new PHGDH inhibitor. The crystal structure of PHGDH in complex with oridonin revealed a new allosteric site. The binding of oridonin to this site reduced the activity of the enzyme by relocating R54, a residue involved in substrate binding. Mutagenesis studies showed that PHGDH activity was very sensitive to cysteine mutations, especially those in the substrate binding domain. Conjugation of oridonin and other reported covalent PHGDH inhibitors to these sites will therefore inhibit PHGDH. In addition to being inhibited enzymatically, PHGDH can also be inhibited by protein aggregation and proteasome-mediated degradation. Several tested PHGDH cancer mutants showed altered enzymatic activity, which can be explained by protein structure and stability. Overall, the above studies present new biophysical and biochemical insights into PHGDH and may facilitate the future design of PHGDH inhibitors.

Improved production of the non-native cofactor F420 in Escherichia coli.

The deazaflavin cofactor F420 is a low-potential, two-electron redox cofactor produced by some Archaea and Eubacteria that is involved in methanogenesis and methanotrophy, antibiotic biosynthesis, and xenobiotic metabolism. However, it is not produced by bacterial strains commonly used for industrial biocatalysis or recombinant protein production, such as Escherichia coli, limiting our ability to exploit it as an enzymatic cofactor and produce it in high yield. Here we have utilized a genome-scale metabolic model of E. coli and constraint-based metabolic modelling of cofactor F420 biosynthesis to optimize F420 production in E. coli. This analysis identified phospho-enol pyruvate (PEP) as a limiting precursor for F420 biosynthesis, explaining carbon source-dependent differences in productivity. PEP availability was improved by using gluconeogenic carbon sources and overexpression of PEP synthase. By improving PEP availability, we were able to achieve a ~ 40-fold increase in the space-time yield of F420 compared with the widely used recombinant Mycobacterium smegmatis expression system. This study establishes E. coli as an industrial F420-production system and will allow the recombinant in vivo use of F420-dependent enzymes for biocatalysis and protein engineering applications.

Metabolic shift to serine biosynthesis through 3-PG accumulation and PHGDH induction promotes tumor growth in pancreatic cancer.

Cancer cells craftily adapt their energy metabolism to their microenvironment. Nutrient deprivation due to hypovascularity and fibrosis is a major characteristic of pancreatic ductal adenocarcinoma (PDAC); thus, PDAC cells must produce energy intrinsically. However, the enhancement of energy production via activating Kras mutations is insufficient to explain the metabolic rewiring of PDAC cells. Here, we investigated the molecular mechanism underlying the metabolic shift in PDAC cells under serine starvation. Amino acid analysis revealed that the concentrations of all essential amino acids and most nonessential amino acids were decreased in the blood of PDAC patients. In addition, the plasma serine concentration was significantly higher in PDAC patients with PHGDH-high tumors than in those with PHGDH-low tumors. Although the growth and tumorigenesis of PK-59 cells with PHGDH promoter hypermethylation were significantly decreased by serine starvation, these activities were maintained in PDAC cell lines with PHGDH promoter hypomethylation by serine biosynthesis through PHGDH induction. In fact, DNA methylation analysis by pyrosequencing revealed that the methylation status of the PHGDH promoter was inversely correlated with the PHGDH expression level in human PDAC tissues. In addition to PHGDH induction by serine starvation, PDAC cells showed enhanced serine biosynthesis under serine starvation through 3-PG accumulation via PGAM1 knockdown, resulting in enhanced PDAC cell growth and tumor growth. However, PHGDH knockdown efficiently suppressed PDAC cell growth and tumor growth under serine starvation. These findings provide evidence that targeting the serine biosynthesis pathway by inhibiting PHGDH is a potent therapeutic approach to eliminate PDAC cells in nutrient-deprived microenvironments.

Heterologous expression of membrane-bound alcohol dehydrogenase-encoding genes for glyceric acid production using Gluconobacter sp. CHM43 and its derivatives.

In contrast to D-glyceric acid (D-GA) production with 99% enantiomeric excess (ee) by Acetobacter tropicalis NBRC 16470, Gluconobacter sp. CHM43 produced 19.6 g L-1 of D-GA with 73.7% ee over 4 days of incubation in flask culture. To investigate the reason for this enantiomeric composition of GA, the genes encoding membrane-bound alcohol dehydrogenase (mADH) of A. tropicalis NBRC 16470, composed of three subunits (adhA, adhB, and adhS), were cloned using the broad-host-range vector pBBR1MCS-2 and heterologously expressed in Gluconobacter sp. CHM43 and its ΔadhAB ΔsldBA derivative TORI4. Reverse-transcription quantitative real-time polymerase chain reaction demonstrated that adhABS genes from A. tropicalis were expressed in TORI4 transformants, and their membrane fraction exhibited mADH activities of 0.13 and 0.31 U/mg with or without AdhS, respectively. Compared with the GA production of TORI4-harboring pBBR1MCS-2 (1.23 g L-1), TORI4 transformants expressing adhABS and adhAB showed elevated GA production of 2.46 and 3.67 g L-1, respectively, suggesting a negative effect of adhS gene expression on GA production as well as mADH activity in TORI4. Although TORI4 was found to produce primarily L-GA with 42.5% ee, TORI4 transformants expressing adhABS and adhAB produced D-GA with 27.6% and 49.0% ee, respectively, demonstrating that mADH of A. tropicalis causes a sharp increase in the enantiomeric composition of D-GA. These results suggest that one reason for D-GA production with 73.7% ee in Gluconobacter spp. might be a property of the host, which possibly produces L-GA intracellularly. KEY POINTS: • Membrane-bound ADH from Acetobacter tropicalis showed activity in Gluconobacter sp. • D-GA production from glycerol was performed using recombinant Gluconobacter sp. • Enantiomeric excess of D-GA was affected by both membrane and intracellular ADHs.

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.

Arabidopsis thaliana 2,3-bisphosphoglycerate-independent phosphoglycerate mutase 2 activity requires serine 82 phosphorylation.

Phosphoglycerate mutases (PGAMs) catalyse the reversible isomerisation of 3-phosphoglycerate and 2-phosphoglycerate, a step of glycolysis. PGAMs can be sub-divided into 2,3-bisphosphoglycerate-dependent (dPGAM) and -independent (iPGAM) enzymes. In plants, phosphoglycerate isomerisation is carried out by cytosolic iPGAM. Despite its crucial role in catabolism, little is known about post-translational modifications of plant iPGAM. In Arabidopsis thaliana, phosphoproteomics analyses have previously identified an iPGAM phosphopeptide where serine 82 is phosphorylated. Here, we show that this phosphopeptide is less abundant in dark-adapted compared to illuminated Arabidopsis leaves. In silico comparison of iPGAM protein sequences and 3D structural modelling of AtiPGAM2 based on non-plant iPGAM enzymes suggest a role for phosphorylated serine in the catalytic reaction mechanism. This is confirmed by the activity (or the lack thereof) of mutated recombinant Arabidopsis iPGAM2 forms, affected in different steps of the reaction mechanism. We thus propose that the occurrence of the S82-phosphopeptide reflects iPGAM2 steady-state catalysis. Based on this assumption, the metabolic consequences of a higher iPGAM activity in illuminated versus darkened leaves are discussed.

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.

Salmonella Typhimurium reprograms macrophage metabolism via T3SS effector SopE2 to promote intracellular replication and virulence.

Salmonella Typhimurium establishes systemic infection by replicating in host macrophages. Here we show that macrophages infected with S. Typhimurium exhibit upregulated glycolysis and decreased serine synthesis, leading to accumulation of glycolytic intermediates. The effects on serine synthesis are mediated by bacterial protein SopE2, a type III secretion system (T3SS) effector encoded in pathogenicity island SPI-1. The changes in host metabolism promote intracellular replication of S. Typhimurium via two mechanisms: decreased glucose levels lead to upregulated bacterial uptake of 2- and 3-phosphoglycerate and phosphoenolpyruvate (carbon sources), while increased pyruvate and lactate levels induce upregulation of another pathogenicity island, SPI-2, known to encode virulence factors. Pharmacological or genetic inhibition of host glycolysis, activation of host serine synthesis, or deletion of either the bacterial transport or signal sensor systems for those host glycolytic intermediates impairs S. Typhimurium replication or virulence.