4,118 research outputs found

    Hydrophobicity and Charge Shape Cellular Metabolite Concentrations

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    What governs the concentrations of metabolites within living cells? Beyond specific metabolic and enzymatic considerations, are there global trends that affect their values? We hypothesize that the physico-chemical properties of metabolites considerably affect their in-vivo concentrations. The recently achieved experimental capability to measure the concentrations of many metabolites simultaneously has made the testing of this hypothesis possible. Here, we analyze such recently available data sets of metabolite concentrations within E. coli, S. cerevisiae, B. subtilis and human. Overall, these data sets encompass more than twenty conditions, each containing dozens (28-108) of simultaneously measured metabolites. We test for correlations with various physico-chemical properties and find that the number of charged atoms, non-polar surface area, lipophilicity and solubility consistently correlate with concentration. In most data sets, a change in one of these properties elicits a ∼100 fold increase in metabolite concentrations. We find that the non-polar surface area and number of charged atoms account for almost half of the variation in concentrations in the most reliable and comprehensive data set. Analyzing specific groups of metabolites, such as amino-acids or phosphorylated nucleotides, reveals even a higher dependence of concentration on hydrophobicity. We suggest that these findings can be explained by evolutionary constraints imposed on metabolite concentrations and discuss possible selective pressures that can account for them. These include the reduction of solute leakage through the lipid membrane, avoidance of deleterious aggregates and reduction of non-specific hydrophobic binding. By highlighting the global constraints imposed on metabolic pathways, future research could shed light onto aspects of biochemical evolution and the chemical constraints that bound metabolic engineering efforts

    The protein cost of metabolic fluxes: prediction from enzymatic rate laws and cost minimization

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    Bacterial growth depends crucially on metabolic fluxes, which are limited by the cell's capacity to maintain metabolic enzymes. The necessary enzyme amount per unit flux is a major determinant of metabolic strategies both in evolution and bioengineering. It depends on enzyme parameters (such as kcat and KM constants), but also on metabolite concentrations. Moreover, similar amounts of different enzymes might incur different costs for the cell, depending on enzyme-specific properties such as protein size and half-life. Here, we developed enzyme cost minimization (ECM), a scalable method for computing enzyme amounts that support a given metabolic flux at a minimal protein cost. The complex interplay of enzyme and metabolite concentrations, e.g. through thermodynamic driving forces and enzyme saturation, would make it hard to solve this optimization problem directly. By treating enzyme cost as a function of metabolite levels, we formulated ECM as a numerically tractable, convex optimization problem. Its tiered approach allows for building models at different levels of detail, depending on the amount of available data. Validating our method with measured metabolite and protein levels in E. coli central metabolism, we found typical prediction fold errors of 3.8 and 2.7, respectively, for the two kinds of data. ECM can be used to predict enzyme levels and protein cost in natural and engineered pathways, establishes a direct connection between protein cost and thermodynamics, and provides a physically plausible and computationally tractable way to include enzyme kinetics into constraint-based metabolic models, where kinetics have usually been ignored or oversimplified

    A quantitative study of the relationships between morphology, physiology and geldanamycin synthesis in submerged cultures of Streptomyces hygroscopicus var. geldanus

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    Microbially produced secondary metabolites such as antibiotics have tremendous economic importance. However, most are produced by filamentous organisms which exhibit diverse growth patterns presenting challenges for industrial fermentation. There are many factors affecting secondary metabolite production which concomitantly impact on morphology, thus it is difficult to distinguish the key driver for productivity. Streptomyces spp. is a genus of filamentous organisms that together synthesise over 4000 bioactive compounds. Streptomyces hygroscopicus var. geldanus produces the secondary metabolite geldanamycin, a novel chemotherapeutic compound, in submerged fermentation. This organism represents an ideal system for experimentation in order to elucidate the relationships between morphology, physiology and secondary metabolite production. The effects of a variety of microbiological (inoculum size), physical (glass beads) and chemical (surfactants, calcium ions, magnesium ions) factors on morphological development were examined as part of this study. Inclusion of the divalent cations magnesium or calcium was demonstrated to alter the cell surface hydrophobicity of the organism, provoking dispersion or aggregation of cells respectively, and stimulating great disparity in geldanamycin yields. Indeed, in all instances, morphology was found to impact considerably on secondary metabolite formation, with smaller pellet sizes optimal for geldanamycin synthesis. Investigation of the respiration rate of Streptomyces hygroscopicus var. geldanus revealed that a linear relationship existed between this parameter and geldanamycin production. Submerged cultures consisting primarily of small pellets, less than 0.5mm in diameter, were more metabolically active and concomitantly produced more geldanamycin. Nonetheless, it was also demonstrated that other explicit factors exist which do not affect morphology or respiration but regulate geldanamycin synthesis through feedback inhibition of the direct metabolic pathway. This study has demonstrated that, in Streptomyces hygroscopicus var. geldanus, the bulk of factors that affect morphology impact significantly on respiration, and it is this parameter that is the key driver of secondary metabolite production. This case study provides new insights into the regulation of geldanamycin production in Streptomyces hygroscopicus var. geldanus and provides a basis for elucidation of the relationships between morphology, physiology and secondary metabolism in other filamentous micro-organisms

    Mass spectrometric characterization of flavonoids and in vitro intestinal transport and bioactivity

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    The thermodynamic landscape of carbon redox biochemistry

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    Redox biochemistry plays a key role in the transduction of chemical energy in all living systems. Observed redox reactions in metabolic networks represent only a minuscule fraction of the space of all possible redox reactions. Here we ask what distinguishes observed, natural redox biochemistry from the space of all possible redox reactions between natural and non-natural compounds. We generate the set of all possible biochemical redox reactions involving linear chain molecules with a fixed numbers of carbon atoms. Using cheminformatics and quantum chemistry tools we analyze the physicochemical and thermodynamic properties of natural and non-natural compounds and reactions. We find that among all compounds, aldose sugars are the ones with the highest possible number of connections (reductions and oxidations) to other molecules. Natural metabolites are significantly enriched in carboxylic acid functional groups and depleted in carbonyls, and have significantly higher solubilities than non-natural compounds. Upon constructing a thermodynamic landscape for the full set of reactions as a function of pH and of steady-state redox cofactor potential, we find that, over this whole range of conditions, natural metabolites have significantly lower energies than the non-natural compounds. For the set of 4-carbon compounds, we generate a Pourbaix phase diagram to determine which metabolites are local energetic minima in the landscape as a function of pH and redox potential. Our results suggest that, across a set of conditions, succinate and butyrate are local minima and would thus tend to accumulate at equilibrium. Our work suggests that metabolic compounds could have been selected for thermodynamic stability, and yields insight into thermodynamic and design principles governing nature’s metabolic redox reactions.https://www.biorxiv.org/content/10.1101/245811v1Othe

    Prediction from Enzymatic Rate Laws and Cost Minimization

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    Bacterial growth depends crucially on metabolic fluxes, which are limited by the cell’s capacity to maintain metabolic enzymes. The necessary enzyme amount per unit flux is a major determinant of metabolic strategies both in evolution and bioengineering. It depends on enzyme parameters (such as kcat and KM constants), but also on metabolite concentrations. Moreover, similar amounts of different enzymes might incur different costs for the cell, depending on enzyme-specific properties such as protein size and half-life. Here, we developed enzyme cost minimization (ECM), a scalable method for computing enzyme amounts that support a given metabolic flux at a minimal protein cost. The complex interplay of enzyme and metabolite concentrations, e.g. through thermodynamic driving forces and enzyme saturation, would make it hard to solve this optimization problem directly. By treating enzyme cost as a function of metabolite levels, we formulated ECM as a numerically tractable, convex optimization problem. Its tiered approach allows for building models at different levels of detail, depending on the amount of available data. Validating our method with measured metabolite and protein levels in E. coli central metabolism, we found typical prediction fold errors of 4.1 and 2.6, respectively, for the two kinds of data. This result from the cost-optimized metabolic state is significantly better than randomly sampled metabolite profiles, supporting the hypothesis that enzyme cost is important for the fitness of E. coli. ECM can be used to predict enzyme levels and protein cost in natural and engineered pathways, and could be a valuable computational tool to assist metabolic engineering projects. Furthermore, it establishes a direct connection between protein cost and thermodynamics, and provides a physically plausible and computationally tractable way to include enzyme kinetics into constraint-based metabolic models, where kinetics have usually been ignored or oversimplified

    Designing organometallic compounds for catalysis and therapy

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    Bioorganometallic chemistry is a rapidly developing area of research. In recent years organometallic compounds have provided a rich platform for the design of effective catalysts, e.g. for olefin metathesis and transfer hydrogenation. Electronic and steric effects are used to control both the thermodynamics and kinetics of ligand substitution and redox reactions of metal ions, especially Ru II. Can similar features be incorporated into the design of targeted organometallic drugs? Such complexes offer potential for novel mechanisms of drug action through incorporation of outer-sphere recognition of targets and controlled activation features based on ligand substitution as well as metal- and ligand-based redox processes. We focus here on η 6-arene, η 5-cyclopentadienyl sandwich and half-sandwich complexes of Fe II, Ru II, Os II and Ir III with promising activity towards cancer, malaria, and other conditions. © 2012 The Royal Society of Chemistry

    The Potential effect of Low Cell Osmolarity on Cell Function through decreased concentration of enzyme substrates

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    © The Author(s) 2018. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. Some freshwater algae have lower (180 osmol m −3 ). Low osmolarities are related to the presence of flagella and the low energy cost of active water efflux following downhill water influx unconstrained by cell walls covering the plasmalemma, and the low resource cost of cell wall synthesis with the same mechanical degree of safety. One consequence of low intracellular osmolarity is limitation on the concentration of metabolites, that is, substrates and products of enzyme activity. Models of the flux through metabolic pathways, and hence the specific growth rate, using steady-state concentrations of enzymes and metabolites have involved organisms with intracellular metabolite osmolarities >280 osmol m −3 , where the metabolite concentrations are much greater than the total osmolarity of some freshwater algae. Since the protein concentration (mol m −3 ) in the cells and the specific growth rates of freshwater cells with low and with higher intracellular osmolarity are highly similar, the models of trade-offs between enzyme and metabolite concentrations for cells with high intracellular osmolarity need modification for cells with low intracellular osmolarity. The soluble free-radical scavenger ascorbate can constitute as little as 0.2% of the low intracellular metabolite concentration (mol m −3 ) of low-intracellular-osmolarity cells

    Development of Long-acting Nanoformulated Abacavir ProTides

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    Combination antiretroviral therapy (cART) for treatment of human immunodeficiency virus (HIV) infection demands life-long regimen adherence. Treatment interruptions lead to a lack of virologic control and the emergence of viral mutations and drug resistance. To address these, our laboratory developed long-acting (LA) parenteral administered nanoformulated abacavir prodrug (NMABC) by formulating myristoylated abacavir (MABC) with poloxamer 407 as stabilizer, which sustained ABC levels for up to 2 weeks with low levels of active drug metabolite (CBV-TP). To more significantly extend the apparent half-life of ABC and improve its antiretroviral activities, we developed second generation long-acting ABC prodrugs by ProTide technologies. This modification can deliver pre-activated nucleoside analog inside the target cells. The work proceeded in defined manner. First, we synthesized then characterized traditional ProTides of ABC, AlaMe, with ProTide technology. The antiretroviral activity (EC50) of AlaMe was 7-fold lower then native ABC. Then, AlaMe loaded and lipid coated PLGA nanoparticle was prepared withna size of 250 nm by thin film hydration (NAlaMe). NAlaMe can be easily taken up and retained by monocyte-derived macrophages (MDM) for weeks and protect MDM against HIV infections. Male BALB/cJ mice were subcutaneously administered 54.3mg/kg ABC equivalents of NAlaMe and ABC. NAlaMe treated mice had higher CBV-TP levels than ABC at 24 and 72 h after administration. These results showed that ProTide technologies can be a useful strategy for the development of second-generation ABC LA. Second, ProTide technology was combined with LA technology. Three ABC ProTides (AlaMe, PheMe and PheC22) were successfully synthesized. AlaMe and PheMe were traditional ProTides. PheC22 possessed a long-chain fatty alcohol moiety. All three ProTides demonstrated improved antiretroviral activities compared to ABC. Nanosuspensions of PheMe (NPheMe) and PheC22 (NPheC22) were formulated using P407 as stabilizer with sizes of 388 and 340 nm, respectively. Cell uptake, retention and antiretroviral activity of NAlaMe, NPheMe, NPheC22 were tested in MDM. NPheC22 had the highest uptake, retention and was able to protect MDM against HIV-1 infection up to 30 days. Combinatorial strategies thus show advantages for the development of second-generation ABC LA.ABC ProTides had long-chain fatty esters were specifically named ABC ProTides LA. Third, we investigated the influence of different combination of amino acid and long-chain fatty alcohol on activities of ABC ProTides LA. Six ABC ProTides LA: AlaC14, AlaC18, AlaC22, PheC14, PheC18 and PheC22 were synthesized and characterized. The EC50 of ABC ProTides were up to 100- or 10-fold lower in MDM or CEM CD4+T cells against HIV infection compared to ABC. CBV-TP was increased by up to 45-fold of 8-fold in MDM or CEM CD4+T cells. Nanosuspension of all six ProTides were formulated using DSPG-mPEG2Kand Tween 80 as stabilizers. The six nanosuspension had sizes range from 220 - 300 nm. Nanosuspensions were taken up, retained and released by MDM and protected MDM against HIV-1 infection more than 28 days. PK studies in rats showed that ProTide nanosuspension provide CBV-TP over a longer time than ABC. NAlaC18 provided clinically relevant CBV-TP levels in rats up to 7 days following a single injection. In conclusion, ProTides and LA technologies were combinationally used to create second generation ABC LA. These works are a step forward in the development of long acting nucleoside reverse transcriptase inhibitors for human use

    The Impact of Oxygen on Metabolic Evolution: A Chemoinformatic Investigation

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    The appearance of planetary oxygen likely transformed the chemical and biochemical makeup of life and probably triggered episodes of organismal diversification. Here we use chemoinformatic methods to explore the impact of the rise of oxygen on metabolic evolution. We undertake a comprehensive comparative analysis of structures, chemical properties and chemical reactions of anaerobic and aerobic metabolites. The results indicate that aerobic metabolism has expanded the structural and chemical space of metabolites considerably, including the appearance of 130 novel molecular scaffolds. The molecular functions of these metabolites are mainly associated with derived aspects of cellular life, such as signal transfer, defense against biotic factors, and protection of organisms from oxidation. Moreover, aerobic metabolites are more hydrophobic and rigid than anaerobic compounds, suggesting they are better fit to modulate membrane functions and to serve as transmembrane signaling factors. Since higher organisms depend largely on sophisticated membrane-enabled functions and intercellular signaling systems, the metabolic developments brought about by oxygen benefit the diversity of cellular makeup and the complexity of cellular organization as well. These findings enhance our understanding of the molecular link between oxygen and evolution. They also show the significance of chemoinformatics in addressing basic biological questions
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