Sugar-phosphate Research Articles

Accurate Determination of Isotopic Abundance of Intracellular Metabolites of Saccharopolysporaerythraea Based on Ultra Performance Liquid Chromatography-Triple Quadrupole Mass Spectrometry

Abstract A method was established for measuring 13C isotopic abundance of intracellular metabolites of Saccharopolysporaerythraea by ultra-high performance liquid chromatography (UPLC)-triple quadrupole mass spectrometry. Under the optimized chromatographic conditions of UPLC, and the appropriate and unique tube lens voltage, collision energy and ion pair, one-to-one method, one-to-many method and SIM method were established for measuring 13C isotopic abundance based on the length of the parent and daughter ions carbon chains and whether the daughter ions contain 13C atoms. The three methods were then applied to measure naturally labeled intracellular metabolite standards and 13C labeled samples. According to the gap between the experimental value and the theoretical value, the best method was found for each metabolite of different characteristics. The results showed that one-to-one method was the most effective for measuring the metabolites of daughter ions not containing 13C atoms represented by sugar phosphates, one-to-many method was the best for measuring the metabolites of both parent and daughter ions containing 13C short carbon chains represented by carboxylic acids, SIM method could played a role in measuring the metabolites of both parent and daughter ions containing 13C long carbon chains represented by coenzyme A. These methods had a good measurement precision and could be applied to the measurement of Saccharopolysporaerythraea intracellular metabolites, which would contribute to consequent study of the metabolic mechanism and the efficient expression of erythromycin.

Deciphering Clostridium metabolism and its responses to bioreactor mass transfer during syngas fermentation

This study used 13C tracers and dynamic labeling to reveal metabolic features (nutrients requirements, pathway delineation and metabolite turnover rates) of Clostridium carboxidivorans P7, a model strain for industrial syngas fermentation, and its implication with bioreactor mass transfer. P7 shows poor activity for synthesizing amino acids (e.g., phenylalanine) and thus, needs rich medium for cell growth. The strain has multiple carbon fixation routes (Wood-Ljungdahl pathway, pyruvate:ferredoxin oxidoreductase reaction and anaplerotic pathways) and Re-citrate synthase (Ccar_06155) was a key enzyme in its tricarboxylic acid cycle (TCA) pathway. High fluxes were observed in P7’s Wood-Ljungdahl pathway, right branch of TCA cycle, pyruvate synthesis, and sugar phosphate pathways, but the cells anabolic pathways were strikingly slow. In bioreactor culture, when syngas flowrate increased from 1 to 10 mL/min, P7 strain produced same amount of total extracellular products (acids and alcohols) but high flowrate favored alcohol accumulation. This observation was due to the mass transfer limitation influencing energy metabolism (CO/H2 oxidation for cofactor generations) more prominently than carbon fixation. When syngas flowrate increased from 10 of 20 mL/min, the alcohol productivity was not improved and the labeling rate (~0.03 h−1) of key metabolite acetyl-CoA reached to P7 strain’s metabolism limitation regime.

Glucose-1-phosphate uridylyltransferase from Erwinia amylovora: Activity, structure and substrate specificity

Erwinia amylovora, a Gram-negative plant pathogen, is the causal agent of Fire Blight, a contagious necrotic disease affecting plants belonging to the Rosaceae family, including apple and pear. E. amylovora is highly virulent and capable of rapid dissemination in orchards; effective control methods are still lacking. One of its most important pathogenicity factors is the exopolysaccharide amylovoran. Amylovoran is a branched polymer made by the repetition of units mainly composed of galactose, with some residues of glucose, glucuronic acid and pyruvate. E. amylovora glucose-1-phosphate uridylyltransferase (UDP-glucose pyrophosphorylase, EC 2.7.7.9) has a key role in amylovoran biosynthesis. This enzyme catalyses the production of UDP-glucose from glucose-1-phosphate and UTP, which the epimerase GalE converts into UDP-galactose, the main building block of amylovoran.We determined EaGalU kinetic parameters and substrate specificity with a range of sugar 1-phosphates. At time point 120min the enzyme catalysed conversion of the sugar 1-phosphate into the corresponding UDP-sugar reached 74% for N-acetyl-α-d-glucosamine 1-phosphate, 28% for α-d-galactose 1-phosphate, 0% for α-d-galactosamine 1-phosphate, 100% for α-d-xylose 1-phosphate, 100% for α-d-glucosamine 1-phosphate, 70% for α-d-mannose 1-phosphate, and 0% for α-d-galacturonic acid 1-phosphate. To explain our results we obtained the crystal structure of EaGalU and augmented our study by docking the different sugar 1-phosphates into EaGalU active site, providing both reliable models for substrate binding and enzyme specificity, and a rationale that explains the different activity of EaGalU on the sugar 1-phosphates used. These data demonstrate EaGalU potential as a biocatalyst for biotechnological purposes, as an alternative to the enzyme from Escherichia coli, besides playing an important role in E. amylovora pathogenicity.

Mechanism of Enzyme Repair by the AAA+ Chaperone Rubisco Activase

How AAA+ chaperones conformationally remodel specific target proteins in an ATP-dependent manner is not well understood. Here, we investigated the mechanism of the AAA+ protein Rubisco activase (Rca) in metabolic repair of the photosynthetic enzyme Rubisco, a complex of eight large (RbcL) and eight small (RbcS) subunits containing eight catalytic sites. Rubisco is prone to inhibition by tight-binding sugar phosphates, whose removal is catalyzed by Rca. We engineered a stable Rca hexamer ring and analyzed its functional interaction with Rubisco. Hydrogen/deuterium exchange and chemical crosslinking showed that Rca structurally destabilizes elements of the Rubisco active site with remarkable selectivity. Cryo-electron microscopy revealed that Rca docks onto Rubisco over one active site at a time, positioning the C-terminal strand of RbcL, which stabilizes the catalytic center, for access to the Rca hexamer pore. The pulling force of Rca is fine-tuned to avoid global destabilization and allow for precise enzyme repair.

Identification of a d-Arabinose-5-Phosphate Isomerase in the Gram-Positive Clostridium tetani.

d-Arabinose-5-phosphate (A5P) isomerases (APIs) catalyze the interconversion of d-ribulose-5-phosphate and d-arabinose-5-phosphate. Various Gram-negative bacteria, such as the uropathogenic Escherichia coli strain CFT073, contain multiple API paralogs (KdsD, GutQ, KpsF, and c3406) that have been assigned various cellular functions. The d-arabinose-5-phosphate formed by these enzymes seems to play important roles in the biosynthesis of lipopolysaccharide (LPS) and group 2 K-antigen capsules, as well as in the regulation of the cellular d-glucitol uptake and uropathogenic infectivity/virulence. The genome of a Gram-positive pathogenic bacterium, Clostridium tetani, contains a gene encoding a putative API, C. tetani API (CtAPI), even though C. tetani lacks both LPS and capsid biosynthetic genes. To better understand the physiological role of d-arabinose-5-phosphate in this Gram-positive organism, recombinant CtAPI was purified and characterized. CtAPI displays biochemical characteristics similar to those of APIs from Gram-negative organisms and complements the API deficiency of an E. coli API knockout strain. Thus, CtAPI represents the first d-arabinose-5-phosphate isomerase to be identified and characterized from a Gram-positive bacterium.IMPORTANCE The genome of Clostridium tetani, a pathogenic Gram-positive bacterium and the causative agent of tetanus, contains a gene (the CtAPI gene) that shares high sequence similarity with those of genes encoding d-arabinose-5-phosphate isomerases. APIs play an important role within Gram-negative bacteria in d-arabinose-5-phosphate production for lipopolysaccharide biosynthesis, capsule formation, and regulation of cellular d-glucitol uptake. The significance of our research is in identifying and characterizing CtAPI, the first Gram-positive API. Our findings show that CtAPI is specific to the interconversion of arabinose-5-phosphate and ribulose-5-phosphate while having no activity with the other sugars and sugar phosphates tested. We have speculated a regulatory role for this API in C. tetani, an organism that does not produce lipopolysaccharide.

Evaluation of acceptor selectivity of Lactococcus lactis ssp. lactis trehalose 6-phosphate phosphorylase in the reverse phosphorolysis and synthesis of a new sugar phosphate.

Trehalose 6-phosphate phosphorylase (TrePP), a member of glycoside hydrolase family 65, catalyzes the reversible phosphorolysis of trehalose 6-phosphate (Tre6P) with inversion of the anomeric configuration to produce β-d-glucose 1-phosphate (β-Glc1P) and d-glucose 6-phosphate (Glc6P). TrePP in Lactococcus lactis ssp. lactis (LlTrePP) is, alongside the phosphotransferase system, involved in the metabolism of trehalose. In this study, recombinant LlTrePP was produced and characterized. It showed its highest reverse phosphorolytic activity at pH 4.8 and 40°C, and was stable in the pH range 5.0-8.0 and at up to 30°C. Kinetic analyses indicated that reverse phosphorolysis of Tre6P proceeded through a sequential bi bi mechanism involving the formation of a ternary complex of the enzyme, β-Glc1P, and Glc6P. Suitable acceptor substrates were Glc6P, and, at a low level, d-mannose 6-phosphate (Man6P). From β-Glc1P and Man6P, a novel sugar phosphate, α-d-Glcp-(1↔1)-α-d-Manp6P, was synthesized with 51% yield.

Surveying the expanding prokaryotic Rubisco multiverse.

The universal, but catalytically modest, CO2-fixing enzyme Rubisco is currently experiencing intense interest by researchers aiming to enhance crop photosynthesis. These efforts are mostly focused on the highly conserved hexadecameric enzyme found in land plants. In comparison, prokaryotic organisms harbor a far greater diversity in Rubisco forms. Recent work towards improving our appreciation of microbial Rubisco properties and harnessing their potential is surveyed. New structural models are providing informative glimpses into catalytic subtleties and diverse oligomeric states. Ongoing characterization is informing us about the conservation of constraints, such as sugar phosphate inhibition and the associated dependence on Rubisco activase helper proteins. Prokaryotic Rubiscos operate under a far wider range of metabolic contexts than the photosynthetic function of higher plant enzymes. Relaxed selection pressures may have resulted in the exploration of a larger volume of sequence space than permitted in organisms performing oxygenic photosynthesis. To tap into the potential of microbial Rubiscos, in vivo selection systems are being used to discover functional metagenomic Rubiscos. Various directed evolution systems to optimize their function have been developed. It is anticipated that this approach will provide access to biotechnologically valuable enzymes that cannot be encountered in the higher plant Rubisco space.

Conformation and electronic structure of DNA in carbon nanotubes: a molecular dynamics and first‐principles study

The conformation and electronic structure of DNA in single-walled carbon nanotubes (SWCNT) were studied by combining molecular dynamics and first-principles methods. It was found that the π orbital of the SWCNT inner wall induces a conformational change in single stranded DNA (ssDNA) and promotes the adsorption of general-sequence oligonucleotides on the inner walls of the SWCNTs due to π–π stacking interactions. Moreover, they found that the adsorbed oligonucleotides form a helix because of electrostatic and torsional interactions within the sugar–phosphate backbone.

Anion-Exchange Chromatography Coupled to High-Resolution Mass Spectrometry: A Powerful Tool for Merging Targeted and Non-targeted Metabolomics

In this work, simultaneous targeted metabolic profiling by isotope dilution and non-targeted fingerprinting is proposed for cancer cell studies. The novel streamlined metabolomics workflow was established using anion-exchange chromatography (IC) coupled to high-resolution mass spectrometry (MS). The separation time of strong anion-exchange (2 mm column, flow rate 380 μL min-1, injection volume 5 μL) could be decreased to 25 min for a target list comprising organic acids, sugars, sugar phosphates, and nucleotides. Internal standardization by fully 13C labeled Pichia pastoris extracts enabled absolute quantification of the primary metabolites in adherent cancer cell models. Limits of detection (LODs) in the low nanomolar range and excellent intermediate precisions of the isotopologue ratios (on average <5%,N = 5, over 40 h) were observed. As a result of internal standardization, linear dynamic ranges over 4 orders of magnitude (5 nM-50 μM, R2 > 0.99) were obtained. Experiments on drug-sensitive versus resistant SW480 cancer cells showed the feasibility of merging analytical tasks into one analytical run. Comparing fingerprinting with and without internal standard proved that the presence of the 13C labeled yeast extract required for absolute quantification was not detrimental to non-targeted data evaluation. Several interesting metabolites were discovered by accurate mass and comparing MS2 spectra (acquired in ddMS2 mode) with spectral libraries. Significant differences revealed distinct metabolic phenotypes of drug-sensitive and resistant SW480 cells.

Nonenzymatic gluconeogenesis-like formation of fructose 1,6-bisphosphate in ice

The evolutionary origins of metabolism, in particular the emergence of the sugar phosphates that constitute glycolysis, the pentose phosphate pathway, and the RNA and DNA backbone, are largely unknown. In cells, a major source of glucose and the large sugar phosphates is gluconeogenesis. This ancient anabolic pathway (re-)builds carbon bonds as cleaved in glycolysis in an aldol condensation of the unstable catabolites glyceraldehyde 3-phosphate and dihydroxyacetone phosphate, forming the much more stable fructose 1,6-bisphosphate. We here report the discovery of a nonenzymatic counterpart to this reaction. The in-ice nonenzymatic aldol addition leads to the continuous accumulation of fructose 1,6-bisphosphate in a permanently frozen solution as followed over months. Moreover, the in-ice reaction is accelerated by simple amino acids, in particular glycine and lysine. Revealing that gluconeogenesis may be of nonenzymatic origin, our results shed light on how glucose anabolism could have emerged in early life forms. Furthermore, the amino acid acceleration of a key cellular anabolic reaction may indicate a link between prebiotic chemistry and the nature of the first metabolic enzymes.

A Non-photosynthetic Diatom Reveals Early Steps of Reductive Evolution in Plastids.

Nonphotosynthetic plastids retain important biological functions and are indispensable for cell viability. However, the detailed processes underlying the loss of plastidal functions other than photosynthesis remain to be fully understood. In this study, we used transcriptomics, subcellular localization, and phylogenetic analyses to characterize the biochemical complexity of the nonphotosynthetic plastids of the apochlorotic diatom Nitzschia sp. NIES-3581. We found that these plastids have lost isopentenyl pyrophosphate biosynthesis and ribulose-1,5-bisphosphate carboxylase/oxygenase-based carbon fixation but have retained various proteins for other metabolic pathways, including amino acid biosynthesis, and a portion of the Calvin-Benson cycle comprised only of glycolysis/gluconeogenesis and the reductive pentose phosphate pathway (rPPP). While most genes for plastid proteins involved in these reactions appear to be phylogenetically related to plastid-targeted proteins found in photosynthetic relatives, we also identified a gene that most likely originated from a cytosolic protein gene. Based on organellar metabolic reconstructions of Nitzschia sp. NIES-3581 and the presence/absence of plastid sugar phosphate transporters, we propose that plastid proteins for glycolysis, gluconeogenesis, and rPPP are retained even after the loss of photosynthesis because they feed indispensable substrates to the amino acid biosynthesis pathways of the plastid. Given the correlated retention of the enzymes for plastid glycolysis, gluconeogenesis, and rPPP and those for plastid amino acid biosynthesis pathways in distantly related nonphotosynthetic plastids and cyanobacteria, we suggest that this substrate-level link with plastid amino acid biosynthesis is a key constraint against loss of the plastid glycolysis/gluconeogenesis and rPPP proteins in multiple independent lineages of nonphotosynthetic algae/plants.

Divergent prebiotic synthesis of pyrimidine and 8-oxo-purine ribonucleotides

Understanding prebiotic nucleotide synthesis is a long standing challenge thought to be essential to elucidating the origins of life on Earth. Recently, remarkable progress has been made, but to date all proposed syntheses account separately for the pyrimidine and purine ribonucleotides; no divergent synthesis from common precursors has been proposed. Moreover, the prebiotic syntheses of pyrimidine and purine nucleotides that have been demonstrated operate under mutually incompatible conditions. Here, we tackle this mutual incompatibility by recognizing that the 8-oxo-purines share an underlying generational parity with the pyrimidine nucleotides. We present a divergent synthesis of pyrimidine and 8-oxo-purine nucleotides starting from a common prebiotic precursor that yields the β-ribo-stereochemistry found in the sugar phosphate backbone of biological nucleic acids. The generational relationship between pyrimidine and 8-oxo-purine nucleotides suggests that 8-oxo-purine ribonucleotides may have played a key role in primordial nucleic acids prior to the emergence of the canonical nucleotides of biology.

Structure-based prediction and identification of 4-epimerization activity of phosphate sugars in class II aldolases

Sugar 4-epimerization reactions are important for the production of rare sugars and their derivatives, which have various potential industrial applications. For example, the production of tagatose, a functional sweetener, from fructose by sugar 4-epimerization is currently constrained because a fructose 4-epimerase does not exist in nature. We found that class II d-fructose-1,6-bisphosphate aldolase (FbaA) catalyzed the 4-epimerization of d-fructose-6-phosphate (F6P) to d-tagatose-6-phosphate (T6P) based on the prediction via structural comparisons with epimerase and molecular docking and the identification of the condensed products of C3 sugars. In vivo, the 4-epimerization activity of FbaA is normally repressed. This can be explained by our results showing the catalytic efficiency of d-fructose-6-phosphate kinase for F6P phosphorylation was significantly higher than that of FbaA for F6P epimerization. Here, we identified the epimerization reactions and the responsible catalytic residues through observation of the reactions of FbaA and l-rhamnulose-1-phosphate aldolases (RhaD) variants with substituted catalytic residues using different substrates. Moreover, we obtained detailed potential epimerization reaction mechanism of FbaA and a general epimerization mechanism of the class II aldolases l-fuculose-1-phosphate aldolase, RhaD, and FbaA. Thus, class II aldolases can be used as 4-epimerases for the stereo-selective synthesis of valuable carbohydrates.

The Small Protein SgrT Controls Transport Activity of the Glucose-Specific Phosphotransferase System.

The bacterial small RNA (sRNA) SgrS has been a fruitful model for discovery of novel RNA-based regulatory mechanisms and new facets of bacterial physiology and metabolism. SgrS is one of only a few characterized dual-function sRNAs. SgrS can control gene expression posttranscriptionally via sRNA-mRNA base-pairing interactions. Its second function is coding for the small protein SgrT. Previous work demonstrated that both functions contribute to relief of growth inhibition caused by glucose-phosphate stress, a condition characterized by disrupted glycolytic flux and accumulation of sugar phosphates. The base-pairing activity of SgrS has been the subject of numerous studies, but the activity of SgrT is less well characterized. Here, we provide evidence that SgrT acts to specifically inhibit the transport activity of the major glucose permease PtsG. Superresolution microscopy demonstrated that SgrT localizes to the cell membrane in a PtsG-dependent manner. Mutational analysis determined that residues in the N-terminal domain of PtsG are important for conferring sensitivity to SgrT-mediated inhibition of transport activity. Growth assays support a model in which SgrT-mediated inhibition of PtsG transport activity reduces accumulation of nonmetabolizable sugar phosphates and promotes utilization of alternative carbon sources by modulating carbon catabolite repression. The results of this study expand our understanding of a basic and well-studied biological problem, namely, how cells coordinate carbohydrate transport and metabolism. Further, this work highlights the complex activities that can be carried out by sRNAs and small proteins in bacteria.IMPORTANCE Sequencing, annotation and investigation of hundreds of bacterial genomes have identified vast numbers of small RNAs and small proteins, the majority of which have no known function. In this study, we explore the function of a small protein that acts in tandem with a well-characterized small RNA during metabolic stress to help bacterial cells maintain balanced metabolism and continue growing. Our results indicate that this protein acts on the glucose transport system, inhibiting its activity under stress conditions in order to allow cells to utilize alternative carbon sources. This work sheds new light on a key biological problem: how cells coordinate carbohydrate transport and metabolism. The study also expands our understanding of the functional capacities of small proteins.

Development of a core Clostridium thermocellum kinetic metabolic model consistent with multiple genetic perturbations

BackgroundClostridium thermocellum is a Gram-positive anaerobe with the ability to hydrolyze and metabolize cellulose into biofuels such as ethanol, making it an attractive candidate for consolidated bioprocessing (CBP). At present, metabolic engineering in C. thermocellum is hindered due to the incomplete description of its metabolic repertoire and regulation within a predictive metabolic model. Genome-scale metabolic (GSM) models augmented with kinetic models of metabolism have been shown to be effective at recapitulating perturbed metabolic phenotypes.ResultsIn this effort, we first update a second-generation genome-scale metabolic model (iCth446) for C. thermocellum by correcting cofactor dependencies, restoring elemental and charge balances, and updating GAM and NGAM values to improve phenotype predictions. The iCth446 model is next used as a scaffold to develop a core kinetic model (k-ctherm118) of the C. thermocellum central metabolism using the Ensemble Modeling (EM) paradigm. Model parameterization is carried out by simultaneously imposing fermentation yield data in lactate, malate, acetate, and hydrogen production pathways for 19 measured metabolites spanning a library of 19 distinct single and multiple gene knockout mutants along with 18 intracellular metabolite concentration data for a Δgldh mutant and ten experimentally measured Michaelis–Menten kinetic parameters.ConclusionsThe k-ctherm118 model captures significant metabolic changes caused by (1) nitrogen limitation leading to increased yields for lactate, pyruvate, and amino acids, and (2) ethanol stress causing an increase in intracellular sugar phosphate concentrations (~1.5-fold) due to upregulation of cofactor pools. Robustness analysis of k-ctherm118 alludes to the presence of a secondary activity of ketol-acid reductoisomerase and possible regulation by valine and/or leucine pool levels. In addition, cross-validation and robustness analysis allude to missing elements in k-ctherm118 and suggest additional experiments to improve kinetic model prediction fidelity. Overall, the study quantitatively assesses the advantages of EM-based kinetic modeling towards improved prediction of C. thermocellum metabolism and develops a predictive kinetic model which can be used to design biofuel-overproducing strains.

Rubisco Activases: AAA+ Chaperones Adapted to Enzyme Repair

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), the key enzyme of the Calvin-Benson-Bassham cycle of photosynthesis, requires conformational repair by Rubisco activase for efficient function. Rubisco mediates the fixation of atmospheric CO2 by catalyzing the carboxylation of the five-carbon sugar ribulose-1,5-bisphosphate (RuBP). It is a remarkably inefficient enzyme, and efforts to increase crop yields by bioengineering Rubisco remain unsuccessful. This is due in part to the complex cellular machinery required for Rubisco biogenesis and metabolic maintenance. To function, Rubisco must undergo an activation process that involves carboxylation of an active site lysine by a non-substrate CO2 molecule and binding of a Mg2+ ion. Premature binding of the substrate RuBP results in an inactive enzyme. Moreover, Rubisco can also be inhibited by a range of sugar phosphates, some of which are “misfire” products of its multistep catalytic reaction. The release of the inhibitory sugar molecule is mediated by the AAA+ protein Rubisco activase (Rca), which couples hydrolysis of ATP to the structural remodeling of Rubisco. Rca enzymes are found in the vast majority of photosynthetic organisms, from bacteria to higher plants. They share a canonical AAA+ domain architecture and form six-membered ring complexes but are diverse in sequence and mechanism, suggesting their convergent evolution. In this review, we discuss recent advances in understanding the structure and function of this important group of client-specific AAA+ proteins.

Cyanobacterial carbon metabolism: Fluxome plasticity and oxygen dependence

Synechocystis sp. strain PCC 6803 has been widely used as a photo-biorefinery chassis. Based on its genome annotation, this species contains a complete TCA cycle, an Embden-Meyerhof-Parnas pathway (EMPP), an oxidative pentose phosphate pathway (OPPP), and an Entner-Doudoroff pathway (EDP). To evaluate how Synechocystis 6803 catabolizes glucose under heterotrophic conditions, we performed 13 C metabolic flux analysis, metabolite pool size analysis, gene knockouts, and heterologous expressions. The results revealed a cyclic mode of flux through the OPPP. Small, but non-zero, fluxes were observed through the TCA cycle and the malic shunt. Independent knockouts of 6-phosphogluconate dehydrogenase (gnd) and malic enzyme (me) corroborated these results, as neither mutant could grow under dark heterotrophic conditions. Our data also indicate that Synechocystis 6803 metabolism relies upon oxidative phosphorylation to generate ATP from NADPH under dark or insufficient light conditions. The pool sizes of intermediates in the TCA cycle, particularly acetyl-CoA, were found to be several fold lower in Synechocystis 6803 (compared to E. coli metabolite pool sizes), while its sugar phosphate intermediates were several-fold higher. Moreover, negligible flux was detected through the native, or heterologous, EDP in the wild type or Δgnd strains under heterotrophic conditions. Comparing photoautotrophic, photomixotrophic, and heterotrophic conditions, the Calvin cycle, OPPP, and EMPP in Synechocystis 6803 possess the ability to regulate their fluxes under various growth conditions (plastic), whereas its TCA cycle always maintains at low levels (rigid). This work also demonstrates how genetic profiles do not always reflect actual metabolic flux through native or heterologous pathways. Biotechnol. Bioeng. 2017;114: 1593-1602. © 2017 Wiley Periodicals, Inc.

Monitoring integrity and localization of modified single-stranded RNA oligonucleotides using ultrasensitive fluorescence methods

Short single-stranded oligonucleotides represent a class of promising therapeutics with diverse application areas. Antisense oligonucleotides, for example, can interfere with various processes involved in mRNA processing through complementary base pairing. Also RNA interference can be regulated by antagomirs, single-stranded siRNA and single-stranded microRNA mimics. The increased susceptibility to nucleolytic degradation of unpaired RNAs can be counteracted by chemical modification of the sugar phosphate backbone. In order to understand the dynamics of such single-stranded RNAs, we investigated their fate after exposure to cellular environment by several fluorescence spectroscopy techniques. First, we elucidated the degradation of four differently modified, dual-dye labeled short RNA oligonucleotides in HeLa cell extracts by fluorescence correlation spectroscopy, fluorescence cross-correlation spectroscopy and Förster resonance energy transfer. We observed that the integrity of the oligonucleotide sequence correlates with the extent of chemical modifications. Furthermore, the data showed that nucleolytic degradation can only be distinguished from unspecific effects like aggregation, association with cellular proteins, or intramolecular dynamics when considering multiple measurement and analysis approaches. We also investigated the localization and integrity of the four modified oligonucleotides in cultured HeLa cells using fluorescence lifetime imaging microscopy. No intracellular accumulation could be observed for unmodified oligonucleotides, while completely stabilized oligonucleotides showed strong accumulation within HeLa cells with no changes in fluorescence lifetime over 24 h. The integrity and accumulation of partly modified oligonucleotides was in accordance with their extent of modification. In highly fluorescent cells, the oligonucleotides were transported to the nucleus. The lifetime of the RNA in the cells could be explained by a balance between release of the oligonucleotides from endosomes, degradation by RNases and subsequent depletion from the cells.

Mechanistic insight into the substrate specificity of 1,2-\u03b2-oligoglucan phosphorylase from Lachnoclostridium phytofermentans

Glycoside phosphorylases catalyze the phosphorolysis of oligosaccharides into sugar phosphates. Recently, we found a novel phosphorylase acting on β-1,2-glucooligosaccharides with degrees of polymerization of 3 or more (1,2-β-oligoglucan phosphorylase, SOGP) in glycoside hydrolase family (GH) 94. Here, we characterized SOGP from Lachnoclostridium phytofermentans (LpSOGP) and determined its crystal structure. LpSOGP is a monomeric enzyme that contains a unique β-sandwich domain (Ndom1) at its N-terminus. Unlike the dimeric GH94 enzymes possessing catalytic pockets at their dimer interface, LpSOGP has a catalytic pocket between Ndom1 and the catalytic domain. In the complex structure of LpSOGP with sophorose, sophorose binds at subsites +1 to +2. Notably, the Glc moiety at subsite +1 is flipped compared with the corresponding ligands in other GH94 enzymes. This inversion suggests the great distortion of the glycosidic bond between subsites −1 and +1, which is likely unfavorable for substrate binding. Compensation for this disadvantage at subsite +2 can be accounted for by the small distortion of the glycosidic bond in the sophorose molecule. Therefore, the binding mode at subsites +1 and +2 defines the substrate specificity of LpSOGP, which provides mechanistic insights into the substrate specificity of a phosphorylase acting on β-1,2-glucooligosaccharides.

Rational design of reversible inhibitors for trehalose 6-phosphate phosphatases

In some organisms, environmental stress triggers trehalose biosynthesis that is catalyzed collectively by trehalose 6-phosphate synthase, and trehalose 6-phosphate phosphatase (T6PP). T6PP catalyzes the hydrolysis of trehalose 6-phosphate (T6P) to trehalose and inorganic phosphate and is a promising target for the development of antibacterial, antifungal and antihelminthic therapeutics. Herein, we report the design, synthesis and evaluation of a library of aryl d-glucopyranoside 6-sulfates to serve as prototypes for small molecule T6PP inhibitors. Steady-state kinetic techniques were used to measure inhibition constants (Ki) of a panel of structurally diverse T6PP orthologs derived from the pathogens Brugia malayi, Ascaris suum, Mycobacterium tuberculosis, Shigella boydii and Salmonella typhimurium. The binding affinities of the most active inhibitor of these T6PP orthologs, 4-n-octylphenyl α-d-glucopyranoside 6-sulfate (9a), were found to be in the low micromolar range. The Ki of 9a with the B. malayi T6PP ortholog is 5.3 ± 0.6 μM, 70-fold smaller than the substrate Michaelis constant. The binding specificity of 9a was demonstrated using several representative sugar phosphate phosphatases from the HAD enzyme superfamily, the T6PP protein fold family of origin. Lastly, correlations drawn between T6PP active site structure, inhibitor structure and inhibitor binding affinity suggest that the aryl d-glucopyranoside 6-sulfate prototypes will find future applications as a platform for development of tailored second-generation T6PP inhibitors.