Production of D-glucaric acid with phosphoglucose isomerase-deficient Saccharomyces cerevisiae.

D-Glucaric acid is a potential biobased platform chemical. Previously mainly Escherichia coli, but also the yeast Saccharomyces cerevisiae, and Pichia pastoris, have been engineered for conversion of D-glucose to D-glucaric acid via myo-inositol. One reason for low yields from the yeast strains is the strong flux towards glycolysis. Thus, to decrease the flux of D-glucose to biomass, and to increase D-glucaric acid yield, the four step D-glucaric acid pathway was introduced into a phosphoglucose isomerase deficient (Pgi1p-deficient) Saccharomyces cerevisiae strain. High D-glucose concentrations are toxic to the Pgi1p-deficient strains, so various feeding strategies and use of polymeric substrates were studied. Uniformly labelled 13C-glucose confirmed conversion of D-glucose to D-glucaric acid. In batch bioreactor cultures with pulsed D-fructose and ethanol provision 1.3 g D-glucaric acid L-1 was produced. The D-glucaric acid titer (0.71 g D-glucaric acid L-1) was lower in nitrogen limited conditions, but the yield, 0.23 g D-glucaric acid [g D-glucose consumed]-1, was among the highest that has so far been reported from yeast. Accumulation of myo-inositol indicated that myo-inositol oxygenase activity was limiting, and that there would be potential to even higher yield. The Pgi1p-deficiency in S. cerevisiae provides an approach that in combination with other reported modifications and bioprocess strategies would promote the development of high yield D-glucaric acid yeast strains.

Deglycation activity of the Escherichia coli glycolytic enzyme phosphoglucose isomerase.

Glycation is a spontaneous chemical reaction, which affects the structure and function of proteins under normal physiological conditions. Therefore, organisms have evolved diverse mechanisms to combat glycation. In this study, we show that the Escherichia coli glycolytic enzyme phosphoglucose isomerase (Pgi) exhibits deglycation activity. We found that E. coli Pgi catalyzes the breakdown of glucose 6-phosphate (G6P)-derived Amadori products (APs) in chicken lysozyme. The affinity of Pgi to the glycated lysozyme (Km, 1.1 mM) was ten times lower than the affinity to its native substrate, fructose 6-phosphate (Km, 0.1 mM). However, the high kinetic constants of the enzyme with the glycated lysozyme (kcat, 396 s-1 and kcat/Km, 3.6 × 105 M-1 s-1) indicated that the Pgi amadoriase activity may have physiological implications. Indeed, when using total E. coli protein (20 mg/mL) as a substrate in the deglycation reaction, we observed a release of G6P from the bacterial protein at a Pgi specific activity of 33 µmol/min/mg. Further, we detected 11.4 % lower APs concentration in protein extracts from Pgi-proficient vs. deficient cells (p = 0.0006) under conditions where the G6P concentration in Pgi-proficient cells was four times higher than in Pgi-deficient cells (p = 0.0001). Altogether, these data point to physiological relevance of the Pgi deglycation activity.

RHBDF1 deficiency suppresses melanoma glycolysis and enhances efficacy of immunotherapy by facilitating glucose-6-phosphate isomerase degradation via TRIM32.

Melanoma is a dangerous form of skin cancer, making it important to investigate new mechanisms and approaches to enhance the effectiveness of treatment. Here, we establish a positive correlation between the human rhomboid family-1 (RHBDF1) protein and melanoma malignancy. We demonstrate that the melanoma RHBDF1 decrease dramatically inhibits tumor growth and the development of lung metastases, which may be related to the impaired glycolysis. We show that RHBDF1 function is essential to the maintenance of high levels of glycolytic enzymes, especially glucose-6-phosphate isomerase (GPI). Additionally, we discover that the E3 ubiquitin ligase tripartite motif-containing 32 (TRIM32) mediates the K27/K63-linked ubiquitination of GPI and the ensuing lysosomal degradation process. We prove that the multi-transmembrane domain of RHBDF1 is in competition with GPI, preventing the latter from interacting with NCL1-HT2A-LIN41 (NHL) domain of TRIM32. We also note that the mouse RHBDF1's R747 and Y799 are crucial for competitive binding and GPI protection. Artificially silencing the Rhbdf1 gene in a mouse melanoma model results in declined lactic acid levels, elevated cytotoxic lymphocyte infiltration, and improved tumor responsiveness to immunotherapy. These results provide credence to the hypothesis that RHBDF1 plays a significant role in melanoma regulation and suggest that blocking RHBDF1 may be an efficient technique for reestablishing the tumor immune microenvironment (TIME) in melanoma and halting its progression.

Preparation of mesoporous silica nanocarriers targeting glucose-6-phosphate isomerase inhibition and application in the treatment of rheumatoid arthritis.

Glucose 6-phosphate isomerase (G6PI) is an indicator to assist in diagnosis of rheumatoid arthritis (RA) and monitor the disease. It also plays a key role in proliferating RA synovial tissues, pannus formation, and invasion and destruction of articular cartilage. In this study, we synthesized nanoparticles targeting G6PI (siG6PI-MSN) using mesoporous silica nanocarriers (MSN) and small interfering RNA (siRNA), followed by identifying the characteristics and functions, and preliminarily exploring their application in the treatment of RA in vivo with a type II collagen-induced arthritis (CIA) rat model. It showed that the synthetic functionalized carrier had a regular pore structure and a specific volume and surface area. No obvious hemolysis or toxicity of the carrier was found when its concentration was below 100 µg/ml. Cytological results in vitro suggested that siG6PI-MSN significantly inhibited G6PI expression and reduced the ability of proliferation, migration, and invasion of FLSs, compared with the siNC-MSN group. In vivo results in the CIA rat model showed that the arthritis index and degree of joint swelling among rats in the siG6PI-MSN-treatment group were significantly lower than those in the control group. Moreover, the number of FLSs in Synovium and the levels of TNF α and IL-1 ß were also significantly decreased in the siG6PI-MSN group. Histopathology of the synovial tissue and cartilage revealed siG6PI-MSN treatment significantly reduced the pathological manifestations of arthritis. In conclusion, siG6PI-MSN effectively suppresses the proliferation and invasive growth of synovial tissue and improve joint swelling and inflammatory infiltration, thereby preventing joint damage in RA. This carrier may be a new therapeutic measure for RA, with potential social and economic benefits.

Cytosolic phosphoglucose isomerase is essential for microsporogenesis and embryogenesis in Arabidopsis.

Phosphoglucose isomerase (PGI) catalyzes the interconversion of fructose-6-phosphate and glucose-6-phosphate, which impacts cell carbon metabolic flow. Arabidopsis (Arabidopsis thaliana) contains two nuclear PGI genes respectively encoding plastidial PGI1 and cytosolic PGI (cPGI). The loss of PGI1 impairs the conversion of F6P of the Calvin-Benson cycle to G6P for the synthesis of transitory starch in leaf chloroplasts. Since cpgi knockout mutants have not yet been obtained, they are thought to be lethal. The cpgi lethality can be rescued by expressing CaMV 35S promoter (p35S)-driven cPGI; however, the complemented line is completely sterile due to pollen degeneration. Here, we generated a cpgi mutant expressing p35S::cPGI-YFP in which YFP fluorescence in developing anthers was undetectable specifically in the tapetum and in pollen, which could be associated with male sterility. We also generated RNAi-cPGI knockdown lines with strong cPGI repression in floral buds that exhibited reduced male fertility due to the degeneration of most pollen. Histological analyses indicated that the synthesis of intersporal callose walls was impaired, causing microsporocytes to fail to separate haploid daughter nuclei to form tetrads, which might be responsible for subsequent pollen degeneration. We successfully isolated cpgi knockout mutants in the progeny of a heterozygous cpgi mutant floral-dipped with sugar solutions. The rescued cpgi mutants exhibited diminished young vegetative growth, reduced female fertility, and impaired intersporal callose wall formation in a meiocyte, and, thus, male sterility. Collectively, our data suggest that cPGI plays a vital role in carbohydrate partitioning, which is indispensable for microsporogenesis and early embryogenesis.

Three amino acid substitutions contributing to thermostability of phosphoglucose isomerase in the Glanville fritillary butterfly.

Temperature is one of the most important environmental factors that affect organisms, especially ectotherms, due to its effects on protein stability. Understanding the general rules that govern thermostability changes in proteins to adapt high-temperature environments is crucial. Here, we report the amino acid substitutions of phosphoglucose isomerase (PGI) related to thermostability in the Glanville fritillary butterfly (Melitaea cinxia, Lepidoptera: Nymphalidae). The PGI encoded by the most common allele in M. cinxia in the Chinese population (G3-PGI), which is more thermal tolerant, is more stable under heat stress than that in the Finnish population (D1-PGI). There are 5 amino acid substitutions between G3-PGI and D1-PGI. Site-directed mutagenesis revealed that the combination of amino acid substitutions of H35Q, M49T, and I64V may increase PGI thermostability. These substitutions alter the 3D structure to increase the interaction between 2 monomers of PGI. Through molecular dynamics simulations, it was found that the amino acid at site 421 is more stable in G3-PGI, confining the motion of the α-helix 420-441 and stabilizing the interaction between 2 PGI monomers. The strategy for high-temperature adaptation through these 3 amino acid substitutions is also adopted by other butterfly species (Boloria eunomia, Aglais urticae, Colias erate, and Polycaena lua) concurrent with M. cinxia in the Tianshan Mountains of China, i.e., convergent evolution in butterflies.

GPI: An indicator for immune infiltrates and prognosis of human breast cancer from a comprehensive analysis.

Glucose-6-phosphate isomerase (GPI) plays an important part in gluconeogenesis and glycolysis through the interconversion of d-glucose-6-phosphate and d-fructose-6-phosphate, and its clinical significance still remains unclear in breast cancer (BRCA). We analyzed the expressions of GPI in BRCA patients to determine prognostic values. Our results showed that the expression levels of GPI were upregulated in BRCA patients, and a high GPI expression is correlated with poor overall survival (OS) in BRCA. At the same time, a high GPI expression is correlated with poor clinicopathological characteristics, such as stage III, over 60 years old, N3, HER2 negative, and estrogen receptor (ER) positive. Further analysis of the influence of GPI on the prognosis of BRCA suggested that 50 genes and 10 proteins were positively correlated with GPI, and these genes and proteins were mainly involved in cell cycle signaling pathways. In addition, in this study, we observed that GPI was closely related to N 6-methyladenosine (m6A) RNA methylation modification and immune cell infiltration and ferroptosis-related gene expression in BRCA, and there was a difference in m6A RNA methylation alterations, immune cell infiltration, and ferroptosis-related gene expression between the high GPI expression group and the low GPI expression group. Finally, we found that GPI in BRCA had 2.6% gene alterations, and BRCA patients with gene alteration of GPI had a poor prognosis in disease-free survival (DFS). Altogether, our work strongly suggested that GPI may serve as a new prognostic biomarker for BRCA patients.

A structural basis for the functional differences between the cytosolic and plastid phosphoglucose isomerase isozymes.

Phosphoglucose isomerase (PGI) catalyzes the interconversion between glucose-6-phosphate (G6P) and fructose-6-phosphate (F6P), thereby regulating sucrose synthesis in plant cells. In general, plants contain a pair of PGI isozymes located in two distinct compartments of the cell (cytosol and plastid) with differences in both the primary structure and the higher-order structure. Previously, we showed that the activity of cytosolic PGI (PGIc) is more robust (activity, thermal stability, substrate turnover rate, etc.) than that of the plastid counterpart (PGIp) in multiple organisms, including wheat, rice, and Arabidopsis. The crystal structures of apoTaPGIc (an isotype cytosol PGIc in Triticum aestivum), TaPGIc-G6P complex, and apoTaPGIp (an isotype plastid PGIp in Triticum aestivum) were first solved in higher plants, especially in crops. In this study, we detailed the structural characteristics related to the biochemical properties and functions of TaPGIs in different plant organelles. We found that the C-terminal domains (CTDs) of TaPGIc and TaPGIp are very different, which affects the stability of the dimerized enzyme, and that Lys213TaPGIc/Lys193TaPGIp and its surrounding residues at the binding pocket gateway may participate in the entrance and exit of substrates. Our findings provide a good example illuminating the evolution of proteins from primary to higher structures as a result of physical barriers and adaptation to the biochemical environment.

Central Sugar Metabolism and the Cell Wall.

The human opportunistic pathogen Aspergillus fumigatus is recognized for its versatile cell wall when it comes to remodeling its components in adaptation to external threats, and this remodeling renders it refractory to antifungals targeting cell wall biosynthesis. A specific role for general sugar metabolism in the regulation of the synthesis of cell wall polymers has been previously demonstrated. Delving deeper into central sugar metabolism may reveal unexpected fundamental aspects in cell wall construction, as shown by the work of Zhou and coworkers (Y. Zhou, K. Yan, Q. Qin, O.G. Raimi, et al., mBio 13:e01426-22, 2022, https://doi.org/10.1128/mbio.01426-22) on the roles of the phosphoglucose isomerase of A. fumigatus in cell wall biosynthesis.

Glucose-6-P/phosphate translocator2 mediates the phosphoglucose-isomerase1-independent response to microbial volatiles.

In Arabidopsis (Arabidopsis thaliana), the plastidial isoform of phosphoglucose isomerase (PGI1) mediates photosynthesis, metabolism, and development, probably due to its involvement in the synthesis of isoprenoid-derived signals in vascular tissues. Microbial volatile compounds (VCs) with molecular masses of <45 Da promote photosynthesis, growth, and starch overaccumulation in leaves through PGI1-independent mechanisms. Exposure to these compounds in leaves enhances the levels of GLUCOSE-6-PHOSPHATE/PHOSPHATE TRANSLOCATOR2 (GPT2) transcripts. We hypothesized that the PGI1-independent response to microbial volatile emissions involves GPT2 action. To test this hypothesis, we characterized the responses of wild-type (WT), GPT2-null gpt2-1, PGI1-null pgi1-2, and pgi1-2gpt2-1 plants to small fungal VCs. In addition, we characterized the responses of pgi1-2gpt2-1 plants expressing GPT2 under the control of a vascular tissue- and root tip-specific promoter to small fungal VCs. Fungal VCs promoted increases in growth, starch content, and photosynthesis in WT and gpt2-1 plants. These changes were substantially weaker in VC-exposed pgi1-2gpt2-1 plants but reverted to WT levels with vascular and root tip-specific GPT2 expression. Proteomic analyses did not detect enhanced levels of GPT2 protein in VC-exposed leaves and showed that knocking out GPT2 reduced the expression of photosynthesis-related proteins in pgi1-2 plants. Histochemical analyses of GUS activity in plants expressing GPT2-GUS under the control of the GPT2 promoter showed that GPT2 is mainly expressed in root tips and vascular tissues around hydathodes. Overall, the data indicated that the PGI1-independent response to microbial VCs involves resetting of the photosynthesis-related proteome in leaves through long-distance GPT2 action.

E74 knockdown represses larval development and chitin synthesis in Hyphantria cunea.

E74 is a key transcription factor induced by 20E, which plays a broad role in many physiological events during insect growth and development, including vitellogenesis, organ remodeling and new tissue formation, programmed cell death and metamorphosis. However, whether it is involved in regulating insect chitin biosynthesis remains largely unclear. Here, the E74 gene was identified for the first time from Hyphantria cunea, a notorious defoliator of forestry. Thereafter, the role of HcE74 in regulating growth, development and chitin synthesis in H. cunea larvae was evaluated. Bioinformatics analysis showed that HcE74 shared the highest identity (95.53%) with E74A of Spodoptera litura, which belonged to Ets superfamily. The results of RNAi bioassay showed that the larval mortality on 6 d after HcE74 knockdown was up to 51.11 ± 6.94%. Meanwhile, a distinct developmental deformity phenotype was found when HcE74 was silenced. These results indicated that HcE74 plays an important role in the development and molting of H. cunea larvae. Moreover, HcE74 knockdown also significantly decreased the expression of four key genes related to chitin synthesis, including glucose-6-phosphate isomerase (HcG6PI), UDP-N-acetylglucosamine pyrophosphorylase (HcUAP), chitin synthetase A (HcCHSA), and chitin synthetase B (HcCHSB). As a result, the content of chitin in midgut and epidermis decreased by 0.54- and 0.08-fold, respectively. Taken together, these results demonstrated that HcE74 not only plays a critical role in the growth and molting of H. cunea larvae, but also probably participates in the transcriptional regulation of genes involved in chitin biosynthesis.

Co-culture with osteoblasts up-regulates glycolysis of chondrocytes through MAPK/HIF-1 pathway.

It is well recognized that the neighbor location between cartilage layer and subchondral bone facilitates the intercellular communication and material exchange. However, the evidence that demonstrates the influence of direct communication between cartilage and subchondral bone on their cell behaviors are still partially unknown. In the current study, we established a co-culture system of chondrocytes and osteoblasts aiming to explore the changes of intracellular metabolism of chondrocytes induced by osteoblasts. By using lactate assay kit, RNA sequencing, qRT-PCR and western blot, we found that osteoblasts enhanced the glycolysis in chondrocytes by characterizing the changes of lactate secretion and cytoplasmic expression, and gene expressions including glucose-6-phosphate isomerase 1 (Gpi1), phosphofructokinase, liver type (Pfkl), lactate dehydrogenase A (Ldha), aldolase, fructose-bisphosphate C (Aldoc), phosphoglycerate kinase 1 (Pgk1), glyceraldehyde-3-phosphate dehydrogenase (Gapdh) and triosephosphate isomerase 1 (Tpi1). The enhanced glycolysis might be due to the activation of HIF-1 signaling and its downstream target, pyruvate dehydrogenase kinase1 (PDK1), by qRT-PCR, western blot and immunofluorescence. We also detected the up-regulation of ERK and p38/MAPK upstream signaling in chondrocytes induced by osteoblasts by western blot and immunofluorescence. The enhanced glycolysis in chondrocytes induced by osteoblasts could help us to better understand the intracellular metabolic mechanism of chondrocytes and cartilage disease occurrence.

Phosphoglucose Isomerase Is Important for Aspergillus fumigatus Cell Wall Biogenesis.

Aspergillus fumigatus is a devastating opportunistic fungal pathogen causing hundreds of thousands of deaths every year. Phosphoglucose isomerase (PGI) is a glycolytic enzyme that converts glucose-6-phosphate to fructose-6-phosphate, a key precursor of fungal cell wall biosynthesis. Here, we demonstrate that the growth of A. fumigatus is repressed by the deletion of pgi, which can be rescued by glucose and fructose supplementation in a 1:10 ratio. Even under these optimized growth conditions, the Δpgi mutant exhibits severe cell wall defects, retarded development, and attenuated virulence in Caenorhabditis elegans and Galleria mellonella infection models. To facilitate exploitation of A. fumigatus PGI as an antifungal target, we determined its crystal structure, revealing potential avenues for developing inhibitors, which could potentially be used as adjunctive therapy in combination with other systemic antifungals. IMPORTANCE Aspergillus fumigatus is an opportunistic fungal pathogen causing deadly infections in immunocompromised patients. Enzymes essential for fungal survival and cell wall biosynthesis are considered potential drug targets against A. fumigatus. PGI catalyzes the second step of the glycolysis pathway, linking glycolysis and the pentose phosphate pathway. As such, PGI has been widely considered as a target for metabolic regulation and therefore a therapeutic target against hypoxia-related diseases. Our study here reveals that PGI is important for A. fumigatus survival and exhibit pleiotropic functions, including development, cell wall glucan biosynthesis, and virulence. We also solved the crystal structure of PGI, thus providing the genetic and structural groundwork for the exploitation of PGI as a potential antifungal target.

Osteoblasts induce glucose-derived ATP perturbations in chondrocytes through noncontact communication.

Cartilage and subchondral bone communicate with each other through material and signal exchanges. However, direct evidence provided by experimental studies on their interactions is insufficient. In the present study, we establish a noncontact co-culture model with a transwell chamber to explore the energetic perturbations in chondrocytes influenced by osteoblasts. Our results indicate that osteoblasts induce more ATP generation in chondrocytes through an energetic shift characterized by enhanced glycolysis and impaired mitochondrial tricarboxylic acid cycle. Enhanced glycolysis is shown by an increase of secreted lactate and the upregulation of glycolytic enzymes, including glucose-6-phosphate isomerase (Gpi), liver type ATP-dependent 6-phosphofructokinase (Pfkl), fructose-bisphosphate aldolase C (Aldoc), glyceraldehyde-3-phosphate dehydrogenase (Gapdh), triosephosphate isomerase (Tpi1), and phosphoglycerate kinase 1 (Pgk1). Impaired mitochondrial tricarboxylic acid cycle is characterized by the downregulation of cytoplasmic aspartate aminotransferase (Got1) and mitochondrial citrate synthase (Cs). Osteoblasts induce the activation of Akt and P38 signaling to mediate ATP perturbations in chondrocytes. This study may deepen our understanding of the maintenance of metabolic homeostasis in the bone-cartilage unit.

Bdellovibrio bacteriovorus phosphoglucose isomerase structures reveal novel rigidity in the active site of a selected subset of enzymes upon substrate binding.

Glycolysis and gluconeogenesis are central pathways of metabolism across all domains of life. A prominent enzyme in these pathways is phosphoglucose isomerase (PGI), which mediates the interconversion of glucose-6-phosphate and fructose-6-phosphate. The predatory bacterium Bdellovibrio bacteriovorus leads a complex life cycle, switching between intraperiplasmic replicative and extracellular 'hunter' attack-phase stages. Passage through this complex life cycle involves different metabolic states. Here we present the unliganded and substrate-bound structures of the B. bacteriovorus PGI, solved to 1.74 Å and 1.67 Å, respectively. These structures reveal that an induced-fit conformational change within the active site is not a prerequisite for the binding of substrates in some PGIs. Crucially, we suggest a phenylalanine residue, conserved across most PGI enzymes but substituted for glycine in B. bacteriovorus and other select organisms, is central to the induced-fit mode of substrate recognition for PGIs. This enzyme also represents the smallest conventional PGI characterized to date and probably represents the minimal requirements for a functional PGI.

Engineering of the cytosolic form of phosphoglucose isomerase into chloroplasts improves plant photosynthesis and biomass.

Starch is the most abundant carbohydrate synthesized in plant chloroplast as the product of photosynthetic carbon assimilation, serving a crucial role in the carbon budget as storage energy. Phosphoglucose isomerase (PGI) catalyzes the interconversion between glucose 6-phosphate (G6P) and fructose 6-phosphate (F6P), which are important metabolic molecules in starch synthesis within chloroplasts and sucrose synthesis in cytosol. Here, we found that the specific activity of recombinantly purified PGI localized in cytosolic PGI (PGIc) was much higher than its plastidic isoenzyme counterpart (PGIp) originated from wheat, rice and Arabidopsis, with wheat PGIc having by far the highest activity. Crystal structures of wheat TaPGIc and TaPGIp proteins were solved and the functional units were homodimers. The active sites of PGIc and PGIp, constituted by the same amino acids, formed different binding pockets. Moreover, PGIc showed slightly lower affinity to the substrate F6P but with much faster turnover rates. Engineering of TaPGIc into chloroplasts of a pgip mutant of Arabidopsis thaliana (atpgip) resulted in starch overaccumulation, increased CO2 assimilation, up to 19% more plant biomass and 27% seed yield productivity. These results show that manipulating starch metabolic pathways in chloroplasts can improve plant biomass and yield productivity.

Phosphodianion Activation of Enzymes for Catalysis of Central Metabolic Reactions.

The activation barriers ΔG⧧ for kcat/Km 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 ß-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.

TM1385 from Thermotoga maritima functions as a phosphoglucose isomerase via cis-enediol-based mechanism with active site redundancy.

Phosphoglucose isomerases (PGIs) belong to a class of enzymes that catalyze the reversible isomerization of glucose-6-phosphate to fructose-6-phosphate. PGIs are crucial in glycolysis and gluconeogenesis pathways and proposed as serving additional extracellular functions in eukaryotic organisms. The phosphoglucose isomerase function of TM1385, a previously uncharacterized protein from Thermotoga maritima, was hypothesized based on structural similarity to established PGI crystal structures and computational docking. Kinetic and colorimetric assays combined with 1H nuclear magnetic resonance (NMR) spectroscopy experimentally confirm that TM1385 is a phosphoglucose isomerase (TmPGI). Evidence of solvent exchange in 1H NMR spectra supports that TmPGI isomerization proceeds through a cis-enediol-based mechanism. To determine which amino acid residues are critical for TmPGI catalysis, putative active site residues were mutated with alanine and screened for activity. Results support that E281 is most important for TmPGI formation of the cis-enediol intermediate, and the presence of either H310 or K422 may be required for catalysis, similar to previous observations from homologous PGIs. However, only TmPGI E281A/Q415A and H310A/K422A double mutations abolished activity, suggesting that there are redundant catalytic residues, and Q415 may participate in sugar phosphate isomerization upon E281 mutation. Combined, we propose that TmPGI E281 participates directly in the cis-enediol intermediate step, and either H310 or K422 may facilitate sugar ring opening and closure.

Constitutive inorganic pyrophosphatase as a reciprocal regulator of three inducible enzymes in Escherichia coli.

BACKGROUND: Previous studies have demonstrated the formation of stable complexes between inorganic pyrophosphatase (PPase) and three other Escherichia coli enzymes - cupin-type phosphoglucose isomerase (cPGI), class I fructose-1,6-bisphosphate aldolase (FbaB) and l-glutamate decarboxylase (GadA). METHODS: Here, we determined by activity measurements how complex formation between these enzymes affects their activities and oligomeric structure. RESULTS: cPGI activity was modulated by all partner proteins, but none was reciprocally affected by cPGI. PPase activity was down-regulated upon complex formation, whereas all other enzymes were up-regulated. For cPGI, the activation was partially counteracted by a shift in dimer ⇆ hexamer equilibrium to inactive hexamer. Complex stoichiometry appeared to be 1:1 in most cases, but FbaB formed both 1:1 and 1:2 complexes with both GadA and PPase, FbaB activation was only observed in the 1:2 complexes. FbaB and GadA induced functional asymmetry (negative kinetic cooperativity) in hexameric PPase, presumably by favoring partial dissociation to trimers. CONCLUSIONS: These four enzymes form all six possible binary complexes in vitro, resulting in modulated activity of at least one of the constituent enzymes. In five complexes, the effects on activity were unidirectional, and in one complex (FbaB⋅PPase), the effects were reciprocal. The effects of potential physiological significance include inhibition of PPase by FbaB and GadA and activation of FbaB and cPGI by PPase. Together, they provide a mechanism for feedback regulation of FbaB and GadA biosynthesis. GENERAL SIGNIFICANCE: These findings indicate the complexity of functionally significant interactions between cellular enzymes, which classical enzymology treats as individual entities, and demonstrate their moonlighting activities as regulators.

Thermodynamics and Kinetics of Glycolytic Reactions. Part II: Influence of Cytosolic Conditions on Thermodynamic State Variables and Kinetic Parameters.

For systems biology, it is important to describe the kinetic and thermodynamic properties of enzyme-catalyzed reactions and reaction cascades quantitatively under conditions prevailing in the cytoplasm. While in part I kinetic models based on irreversible thermodynamics were tested, here in part II, the influence of the presumably most important cytosolic factors was investigated using two glycolytic reactions (i.e., the phosphoglucose isomerase reaction (PGI) with a uni-uni-mechanism and the enolase reaction with an uni-bi-mechanism) as examples. Crowding by macromolecules was simulated using polyethylene glycol (PEG) and bovine serum albumin (BSA). The reactions were monitored calorimetrically and the equilibrium concentrations were evaluated using the equation of state ePC-SAFT. The pH and the crowding agents had the greatest influence on the reaction enthalpy change. Two kinetic models based on irreversible thermodynamics (i.e., single parameter flux-force and two-parameter Noor model) were applied to investigate the influence of cytosolic conditions. The flux-force model describes the influence of cytosolic conditions on reaction kinetics best. Concentrations of magnesium ions and crowding agents had the greatest influence, while temperature and pH-value had a medium influence on the kinetic parameters. With this contribution, we show that the interplay of thermodynamic modeling and calorimetric process monitoring allows a fast and reliable quantification of the influence of cytosolic conditions on kinetic and thermodynamic parameters.