Genetic Analysis of Two Novel GPI Variants Disrupting H Bonds and Localization Characteristics of 55 Gene Variants Associated with Glucose-6-phosphate Isomerase Deficiency.

OBJECTIVE: Glucose-6-phosphate isomerase (GPI) deficiency is a rare hereditary nonspherocytic hemolytic anemia caused by GPI gene variants. This disorder exhibits wide heterogeneity in its clinical manifestations and molecular characteristics, often posing challenges for precise diagnoses using conventional methods. To this end, this study aimed to identify the novel variants responsible for GPI deficiency in a Chinese family. METHODS: The clinical manifestations of the patient were summarized and analyzed for GPI deficiency phenotype diagnosis. Novel compound heterozygous variants of the GPI gene, c.174C>A (p.Asn58Lys) and c.1538G>T (p.Trp513Leu), were identified using whole-exome and Sanger sequencing. The AlphaFold program and Chimera software were used to analyze the effects of compound heterozygous variants on GPI structure. RESULTS: By characterizing 53 GPI missense/nonsense variants from previous literature and two novel missense variants identified in this study, we found that most variants were located in exons 3, 4, 12, and 18, with a few localized in exons 8, 9, and 14. This study identified novel compound heterozygous variants associated with GPI deficiency. These pathogenic variants disrupt hydrogen bonds formed by highly conserved GPI amino acids. CONCLUSION: Early family-based sequencing analyses, especially for patients with congenital anemia, can help increase diagnostic accuracy for GPI deficiency, improve child healthcare, and enable genetic counseling.

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.

A Novel Glucose-6-Phosphate Isomerase Exists in Chicken Breast Meat: A Selenium-Containing Enzyme that Should Be Re-recognized Through New Eyes.

Glucose-6-phosphate isomerase (GPI) is a highly conserved glycolytic enzyme in nature, and less information was available for GPI from hens. In this study a newly discovered selenocysteine (Sec)-containing GPI in common chicken breast meat was first isolated, purified and identified. Data about LC-MS/MS, FTIR and Se species analyses show that the molecular weight of the enzyme is 62,091 Da and only one Sec is inserted at the 403rd position in the highly conserved primary domain SIS_PGI with sugar conversion function. The enzyme shows excellent activity against hydroxyl radicals as vitamin C (Vc) in vitro. It is deduced that the Sec-containing GPI in the chicken meat may depend on Sec in its molecular structure to resist reactive oxygen species (ROS) stress produced by the accompanying biochemical reactions in cells, to protect its stability and maintain its efficient function that catalyzes the conversion of glucose-6-phosphate to fructose-6-phosphate in the critical glycolytic pathway.

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.

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.

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.

Characterization of P. aeruginosa Glucose 6- Phosphate Isomerase: A Functional Insight via In-Vitro Activity Study.

BACKGROUND: Glucose-6-phosphate isomerase (G6PI) catalyses the second step in glycolysis in the reversible interconversion of an aldohexose glucose 6-phosphate, a six membered ring moiety to a ketohexose, fructose 6-phosphate five membered ring moiety. This enzyme is of utmost importance due to its multifunctional role like neuroleukin, autocrine motility factor, etc. in various species. G6PI from Pseudomonas aeruginosa is less explored for its moonlighting properties. These properties can be predicted by studying the active site conservation of residues and their interaction with the specific ligand. METHODS: Here, we study the G6PI in a self-inducible construct in bacterial expression system with its purification using Ni-NTA chromatography. The secondary structure of pure G6PI is estimated using circular dichroism to further predict the proper folding form of the protein. The bioactivity of the purified enzyme is quantified using phosphoglucose isomerase colorimetric kit with a value of 12.5 mU/mL. Differential scanning fluorimetry and isothermal titration calorimetry were employed to monitor the interaction of G6PI with its competitive inhibitor, erythrose 4-phosphate and calculated the Tm, Kd and IC50 values. Further, the homology model for the protein was prepared to study the interaction with the erythrose 4-phosphate. MD simulation of the complex was performed at 100 ns to identify the binding interactions. RESULTS: We identified hydrogen bonds and water bridges dominating the interactions in the active site holding the protein and ligand with strong affinity. CONCLUSION: G6PI was successfully crystallized and data has been collected at 6Å. We are focused on improving the crystal quality for obtaining higher resolution data.

Calibration of the dianionic phosphate group: Validation on the recognition site of the homodimeric enzyme phosphoglucose isomerase.

We calibrate and validate the parameters necessary to represent the dianionic phosphate group (DPG) in molecular mechanics. DPG is an essential fragment of signaling biological molecules and protein-binding ligands. It is a constitutive fragment of biosensors, which bind to the dimer interface of phosphoglucose isomerase (PGI), an intracellular enzyme involved in sugar metabolism, as well as an extracellular protein known as autocrine motility factor (AMF) closely related to metastasis formation. Our long-term objective is to design DPG-based biosensors with enhanced affinities for AMF/PGI cancer biomarker in blood. Molecular dynamics with polarizable potentials could be used toward this aim. This requires to first evaluate the accuracy of such potentials upon representing the interactions of DPG with its PGI ligands and tightly bound water molecules. Such evaluations are done by comparisons with high-level ab initio quantum chemistry (QC) calculations. We focus on the Sum of Interactions Between Fragments Ab initio computed (SIBFA) polarizable molecular mechanics procedure. We present first the results of the DPG calibration. This is followed by comparisons between ΔE(SIBFA) and ΔE(QC) regarding bi-molecular complexes of DPG with the main-chain and side-chain PGI residues, which bind to it in the recognition site. We then consider DPG complexes with an increasing number of PGI residues. The largest QC complexes encompass the entirety of the recognition site, with six structural water molecules totaling up to 211 atoms. A persistent and satisfactory agreement could be shown between ΔE(SIBFA) and ΔE(QC). These validations constitute an essential first step toward large-scale molecular dynamics simulations of DPG-based biosensors bound at the PGI dimer interface. © 2020 Wiley Periodicals, Inc.

Distant Non-Obvious Mutations Influence the Activity of a Hyperthermophilic Pyrococcusfuriosus Phosphoglucose Isomerase.

The cupin-type phosphoglucose isomerase (PfPGI) from the hyperthermophilic archaeon Pyrococcus furiosus catalyzes the reversible isomerization of glucose-6-phosphate to fructose-6-phosphate. We investigated PfPGI using protein-engineering bioinformatics tools to select functionally-important residues based on correlated mutation analyses. A pair of amino acids in the periphery of PfPGI was found to be the dominant co-evolving mutation. The position of these selected residues was found to be non-obvious to conventional protein engineering methods. We designed a small smart library of variants by substituting the co-evolved pair and screened their biochemical activity, which revealed their functional relevance. Four mutants were further selected from the library for purification, measurement of their specific activity, crystal structure determination, and metal cofactor coordination analysis. Though the mutant structures and metal cofactor coordination were strikingly similar, variations in their activity correlated with their fine-tuned dynamics and solvent access regulation. Alternative, small smart libraries for enzyme optimization are suggested by our approach, which is able to identify non-obvious yet beneficial mutations.

First Nonphosphorylated Inhibitors of Phosphoglucose Isomerase Identified by Chemical Library Screening.

Human African trypanosomiasis, Chagas disease, and leishmaniasis are human infections caused by kinetoplastid parasites of the genera Trypanosoma and Leishmania. Besides their severity and global impact, treatments are still challenging. Currently available drugs have important limitations, highlighting the urgent need to develop new drugs. Phosphoglucose isomerase (PGI) is considered a promising target for the development of antiparasitic drugs, as it acts on two essential metabolic pathways, glycolysis and gluconeogenesis. Herein, we describe the identification of new nonphosphorylated inhibitors of Leishmania mexicana PGI ( LmPGI), with the potential for the development of antiparasitic drugs. A fluorescence-based high-throughput screening (HTS) assay was developed by coupling the activities of recombinant LmPGI with glucose-6-phosphate dehydrogenase and diaphorase. This coupled assay was used to screen 42,720 compounds from ChemBridge and TimTec commercial libraries. After confirmatory assays, selected LmPGI inhibitors were tested against homologous Trypanosoma cruzi and humans. The PGI hits are effective against trypanosomatid PGIs, with IC50 values in the micromolar range, and also against the human homologous enzyme. A computational analysis of cavities present on PGI's crystallographic structure suggests a potential binding site for the proposed mixed-type inhibition mechanism.

Substrate-driven chemotactic assembly in an enzyme cascade.

Enzymatic catalysis is essential to cell survival. In many instances, enzymes that participate in reaction cascades have been shown to assemble into metabolons in response to the presence of the substrate for the first enzyme. However, what triggers metabolon formation has remained an open question. Through a combination of theory and experiments, we show that enzymes in a cascade can assemble via chemotaxis. We apply microfluidic and fluorescent spectroscopy techniques to study the coordinated movement of the first four enzymes of the glycolysis cascade: hexokinase, phosphoglucose isomerase, phosphofructokinase and aldolase. We show that each enzyme independently follows its own specific substrate gradient, which in turn is produced by the preceding enzymatic reaction. Furthermore, we find that the chemotactic assembly of enzymes occurs even under cytosolic crowding conditions.

Two novel mutations (p.(Ser160Pro) and p.(Arg472Cys)) causing glucose-6-phosphate isomerase deficiency are associated with erythroid dysplasia and inappropriately suppressed hepcidin.

Glucose-6-phosphate isomerase (GPI) deficiency, a genetic disorder responsible for chronic nonspherocytic hemolytic anemia, is the second most common red blood cell glycolytic enzymopathy. We report three patients from two unrelated families of Czech and Slovak origin with macrocytic hemolytic anemia due to GPI deficiency. The first patient had 15% of residual GPI activity resulting from two new heterozygous missense mutations c.478T>C and c.1414C>T leading to substitutions p.(Ser160Pro) and p.(Arg472Cys). Two other patients (siblings) inherited the same c.1414C>T p.(Arg472Cys) mutation in a homozygous constitution and lost approximately 89% of their GPI activity. Erythroid hyperplasia with dysplastic features was observed in the bone marrow of all three patients. Low hepcidin/ferritin ratio and elevated soluble transferrin receptor detected in our GPI-deficient patients suggest disturbed balance between erythropoiesis and iron metabolism contributing to iron overload.

Cutting Edge: Processing of Oxidized Peptides in Macrophages Regulates T Cell Activation and Development of Autoimmune Arthritis.

APCs are known to produce NADPH oxidase (NOX) 2-derived reactive oxygen species; however, whether and how NOX2-mediated oxidation affects redox-sensitive immunogenic peptides remains elusive. In this study, we investigated a major immunogenic peptide in glucose-6-phosphate isomerase (G6PI), a potential autoantigen in rheumatoid arthritis, which can form internal disulfide bonds. Ag presentation assays showed that presentation of this G6PI peptide was more efficient in NOX2-deficient (Ncf1m1J/m1J mutant) mice, compared with wild-type controls. IFN-γ-inducible lysosomal thiol reductase (GILT), which facilitates disulfide bond-containing Ag processing, was found to be upregulated in macrophages from Ncf1 mutant mice. Ncf1 mutant mice exhibited more severe G6PI peptide-induced arthritis, which was accompanied by the increased GILT expression in macrophages and enhanced Ag-specific T cell responses. Our results show that NOX2-dependent processing of the redox-sensitive autoantigens by APCs modify T cell activity and development of autoimmune arthritis.

Suppression of glucose-6-phosphate-isomerase induced arthritis by oral administration of transgenic rice seeds expressing altered peptide ligands of glucose-6-phosphate-isomerase.

OBJECTIVE: To investigate the effects of transgenic rice seeds expressing the altered peptide ligand (APL) of human glucose-6-phosphate-isomerase (hGPI325-339) in mice model of GPI-induced arthritis (GIA). METHODS: We generated transgenic rice expressing T-cell epitope of hGPI325-339 and APL12 contained in the seed endosperm. The transgenic rice seeds were orally administered prophylactically before the induction of GIA. The severity of arthritis and titers of serum anti-GPI antibodies were evaluated. We examined for IL-17 production in splenocytes and inguinal lymph node (iLN) cells, and analyzed the expression levels of functional molecules in splenocytes. RESULTS: Prophylactic treatment of GIA mice with APL12 transgenic (APL12-TG) rice seeds significantly reduced the severity of arthritis and titers of serum anti-GPI antibodies compared with non-transgenic (Non-TG) rice-treated mice. APL12-TG and hGPI325-339 transgenic (hGPI325-339-TG) rice seeds improved the histopathological arthritis scores and decreased IL-17 production compared with non-TG rice-treated mice. APL12-TG rice-treated GIA mice showed upregulation of Foxp3 and GITR protein in CD4 + CD25 + Foxp3+ cells in the spleen compared with non-TG rice- and hGPI325-339-TG rice-treated mice. CONCLUSION: APL12-TG rice seeds improved the severity of GIA through a decrease in production of IL-17 and anti-GPI antibodies via upregulation of Foxp3 and GITR expression on Treg cells in spleen.

Bioproduction of D-Tagatose from D-Galactose Using Phosphoglucose Isomerase from Pseudomonas aeruginosa PAO1.

Pseudomonas aeruginosa PAO1 phosphoglucose isomerase was purified as an active soluble form by a single-step purification using Ni-NTA chromatography that showed homogeneity on SDS-PAGE with molecular mass ∼62 kDa. The optimum temperature and pH for the maximum isomerization activity with D-galactose were 60 °C and 7.0, respectively. Generally, sugar phosphate isomerases show metal-independent activity but PA-PGI exhibited metal-dependent isomerization activity with aldosugars and optimally catalyzed the D-galactose isomerization in the presence of 1.0 mM MnCl2. The apparent Km and Vmax for D-galactose under standardized conditions were calculated to be 1029 mM (±31.30 with S.E.) and 5.95 U/mg (±0.9 with S.E.), respectively. Equilibrium reached after 180 min with production of 567.51 µM D-tagatose from 1000 mM of D-galactose. Though, the bioconversion ratio is low but it can be increased by immobilization and enzyme engineering. Although various L-arabinose isomerases have been characterized for bioproduction of D-tagatose, P. aeruginosa glucose phosphate isomerase is distinguished from the other L-arabinose isomerases by its optimal temperature (60 °C) for D-tagatose production being mesophilic bacteria, making it an alternate choice for bulk production.

Molecular association of glucose-6-phosphate isomerase and pyruvate kinase M2 with glyceraldehyde-3-phosphate dehydrogenase in cancer cells.

BACKGROUND: For a long time cancer cells are known for increased uptake of glucose and its metabolization through glycolysis. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key regulatory enzyme of this pathway and can produce ATP through oxidative level of phosphorylation. Previously, we reported that GAPDH purified from a variety of malignant tissues, but not from normal tissues, was strongly inactivated by a normal metabolite, methylglyoxal (MG). Molecular mechanism behind MG mediated GAPDH inhibition in cancer cells is not well understood. METHODS: GAPDH was purified from Ehrlich ascites carcinoma (EAC) cells based on its enzymatic activity. GAPDH associated proteins in EAC cells and 3-methylcholanthrene (3MC) induced mouse tumor tissue were detected by mass spectrometry analysis and immunoprecipitation (IP) experiment, respectively. Interacting domains of GAPDH and its associated proteins were assessed by in silico molecular docking analysis. Mechanism of MG mediated GAPDH inactivation in cancer cells was evaluated by measuring enzyme activity, Circular dichroism (CD) spectroscopy, IP and mass spectrometry analyses. RESULT: Here, we report that GAPDH is associated with glucose-6-phosphate isomerase (GPI) and pyruvate kinase M2 (PKM2) in Ehrlich ascites carcinoma (EAC) cells and also in 3-methylcholanthrene (3MC) induced mouse tumor tissue. Molecular docking analyses suggest C-terminal domain preference for the interaction between GAPDH and GPI. However, both C and N termini of PKM2 might be interacting with the C terminal domain of GAPDH. Expression of both PKM2 and GPI is increased in 3MC induced tumor compared with the normal tissue. In presence of 1 mM MG, association of GAPDH with PKM2 or GPI is not perturbed, but the enzymatic activity of GAPDH is reduced to 26.8 ± 5 % in 3MC induced tumor and 57.8 ± 2.3 % in EAC cells. Treatment of MG to purified GAPDH complex leads to glycation at R399 residue of PKM2 only, and changes the secondary structure of the protein complex. CONCLUSION: PKM2 may regulate the enzymatic activity of GAPDH. Increased enzymatic activity of GAPDH in tumor cells may be attributed to its association with PKM2 and GPI. Association of GAPDH with PKM2 and GPI could be a signature for cancer cells. Glycation at R399 of PKM2 and changes in the secondary structure of GAPDH complex could be one of the mechanisms by which GAPDH activity is inhibited in tumor cells by MG.

Plastidic phosphoglucose isomerase is an important determinant of starch accumulation in mesophyll cells, growth, photosynthetic capacity, and biosynthesis of plastidic cytokinins in Arabidopsis.

Phosphoglucose isomerase (PGI) catalyzes the reversible isomerization of glucose-6-phosphate and fructose-6-phosphate. It is involved in glycolysis and in the regeneration of glucose-6-P molecules in the oxidative pentose phosphate pathway (OPPP). In chloroplasts of illuminated mesophyll cells PGI also connects the Calvin-Benson cycle with the starch biosynthetic pathway. In this work we isolated pgi1-3, a mutant totally lacking pPGI activity as a consequence of aberrant intron splicing of the pPGI encoding gene, PGI1. Starch content in pgi1-3 source leaves was ca. 10-15% of that of wild type (WT) leaves, which was similar to that of leaves of pgi1-2, a T-DNA insertion pPGI null mutant. Starch deficiency of pgi1 leaves could be reverted by the introduction of a sex1 null mutation impeding ß-amylolytic starch breakdown. Although previous studies showed that starch granules of pgi1-2 leaves are restricted to both bundle sheath cells adjacent to the mesophyll and stomata guard cells, microscopy analyses carried out in this work revealed the presence of starch granules in the chloroplasts of pgi1-2 and pgi1-3 mesophyll cells. RT-PCR analyses showed high expression levels of plastidic and extra-plastidic ß-amylase encoding genes in pgi1 leaves, which was accompanied by increased ß-amylase activity. Both pgi1-2 and pgi1-3 mutants displayed slow growth and reduced photosynthetic capacity phenotypes even under continuous light conditions. Metabolic analyses revealed that the adenylate energy charge and the NAD(P)H/NAD(P) ratios in pgi1 leaves were lower than those of WT leaves. These analyses also revealed that the content of plastidic 2-C-methyl-D-erythritol 4-phosphate (MEP)-pathway derived cytokinins (CKs) in pgi1 leaves were exceedingly lower than in WT leaves. Noteworthy, exogenous application of CKs largely reverted the low starch content phenotype of pgi1 leaves. The overall data show that pPGI is an important determinant of photosynthesis, energy status, growth and starch accumulation in mesophyll cells likely as a consequence of its involvement in the production of OPPP/glycolysis intermediates necessary for the synthesis of plastidic MEP-pathway derived hormones such as CKs.

Prediction of distal residue participation in enzyme catalysis.

A scoring method for the prediction of catalytically important residues in enzyme structures is presented and used to examine the participation of distal residues in enzyme catalysis. Scores are based on the Partial Order Optimum Likelihood (POOL) machine learning method, using computed electrostatic properties, surface geometric features, and information obtained from the phylogenetic tree as input features. Predictions of distal residue participation in catalysis are compared with experimental kinetics data from the literature on variants of the featured enzymes; some additional kinetics measurements are reported for variants of Pseudomonas putida nitrile hydratase (ppNH) and for Escherichia coli alkaline phosphatase (AP). The multilayer active sites of P. putida nitrile hydratase and of human phosphoglucose isomerase are predicted by the POOL log ZP scores, as is the single-layer active site of P. putida ketosteroid isomerase. The log ZP score cutoff utilized here results in over-prediction of distal residue involvement in E. coli alkaline phosphatase. While fewer experimental data points are available for P. putida mandelate racemase and for human carbonic anhydrase II, the POOL log ZP scores properly predict the previously reported participation of distal residues.

A novel glucose 6-phosphate isomerase from Listeria monocytogenes.

D-Arabinose 5-phosphate isomerases (APIs) catalyze the interconversion of D-ribulose 5-phosphate and D-arabinose 5-phosphate (A5P). A5P is an intermediate in the biosynthesis of 3-deoxy-D-manno-octulosonate (Kdo), an essential component of lipopolysaccharide, the lipopolysaccharide found in the outer membrane of Gram-negative bacteria. The genome of the Gram-positive pathogen Listeria monocytogenes contains a gene encoding a putative sugar isomerase domain API, Q723E8, with significant similarity to c3406, the only one of four APIs from Escherichia coli CFT073 that lacks a cystathionine-ß-synthase domain. However, L. monocytogenes lacks genes encoding any of the other enzymes of the Kdo biosynthesis pathway. Realizing that the discovery of an API in a Gram-positive bacterium could provide insight into an alternate physiological role of A5P in the cell, we prepared and purified recombinant Q723E8. We found that Q723E8 does not possess API activity, but instead is a novel GPI (D-glucose 6-phosphate isomerase). However, the GPI activity of Q723E8 is weak compared with previously described GPIS. L. monocytogenes contains an ortholog of the well-studied two-domain bacterial GPI, so this maybe redundant. Based on this evidence glucose utilization is likely not the primary physiological role of Q723E8.