Interpretation of metabolic flux maps by limitation potentials and constrained limitation sensitivities

Two new concepts, "Limitation Potential" and "Constraint Limitation Sensitivity" are introduced that use definitions derived from metabolic flux analysis (MFA) and metabolic network analysis (MNA). They are applied to interpret a measured flux distribution in the context of all possible flux distributions and thus combine MFA with MNA. The proposed measures are used to quantify and compare the influence of intracellular fluxes on the production yield. The methods are purely based on the stoichiometry of the network and constraints that are given from irreversible fluxes. In contrast to metabolic control analysis (MCA), within this approach no information about the kinetic mechanisms are needed. A limitation potential (LP) is defined as the reduction of the reachable (theoretical) maximum by a measured flux. Measured fluxes that strongly narrow the reachable maximum are assumed to be limiting as the network has no ability to counterbalance the restriction due to the observed flux. In a second step, the sensitivity of the reduced maximum is regarded. This measure provides information about the necessitated changes to reach higher yields. The methods are applied to interpret the capabilities of a network based on measured fluxes for a L-phenylalanine producer. The strain was examined by a series of experiments and three flux maps of the production phase are analyzed. It can be shown that the reachable yield is drastically reduced by the measured efflux into the TCA cycle, while the oxidative pentose-phosphate pathway only plays a secondary role on the reachable maximum.     

Scope and limitation of side‐chain assisted ligation

Side‐chain assisted ligation is an auxiliary‐mediated ligation strategy in which a thiol bearing cyclohexane or cyclopentane is attached to the side‐chain of Asp, Glu, Ser or Thr to function in a similar manner to Cys in a native chemical ligation. Following the ligation step, the auxiliary is removed, without product isolation, under alkaline conditions. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Inhibitors and activators of ADP-ribosylation reactions

ADP-ribosylation reaction, that is the transfer of the ADP-ribose moiety of NAD⁺ to acceptor protein, is catalyzed by two classes of ADP-ribosyltransferases,i.e., poly(ADP-ribose) synthetase and mono (ADP-ribosyl)transferases. These two types differ not only in the number of transferring ADP-ribose units but also in the acceptor amino acid(s) and protein. Their in hibitors, particularly those of poly(ADP-ribose) synthetase, have been successfully employed in studies on biological functions of the enzymes and other related fields of research. Recently, we found many potent and specific inhibitors of poly-(ADP-ribose) synthetase, and broadened their chemical as well as biochemical variety. More recently, we found several potent inhibitors of arginine-specific mono(ADP-ribosyl)transferases and activators of poly(ADP-ribose) synthetase.     

Substrate and inhibitor specificity of 3-hydroxy-3-methylglutaryl-CoA reductase determined with substrate-analogue CoA-thioesters and CoA-thioethers.

1) Analogues of 3-hydroxy-3-methylglutaryl-CoA were prepared in which the substituents at C-3 of the acyl residue were altered. The same analogues were additionally modified by replacement of the thioester oxygen by hydrogen to yield reduction-resistant CoA-thioethers. The interaction of both types of CoA derivatives with a 58-kDa catalytic fragment of human 3-hydroxy-3-methylglutaryl-CoA reductase was studied. 2) This enzyme reduces glutaryl-CoA at a very low rate whereas 3-hydroxyglutaryl-CoA is well reduced, the maximal rate of reduction being 7% that of the physiological substrate. Only half of total 3-hydroxyglutaryl-CoA was attacked, thus reflecting the stereo-specificity of the enzyme for (3S)-3-hydroxy-3-methylglutaryl-CoA. The results invalidate the hitherto assumed absolute substrate specificity of the enzyme. 3) The affinity of both 3-hydroxyglutaryl-CoA and its thioether variant S-(4-carboxy-3-hydroxybutyl)CoA to the reductase, Ki = 0.3 microM and Ki = 0.4 microM, respectively, is higher than that of the physiological substrate, Km = 1.5 microM (data related to (S)-diastereomer). The results show for the first time that the methyl-group effect observed with the inhibitor lovastatin is an intrinsic property of the enzyme. 4) All of the prepared CoA derivatives are purely competitive inhibitors of the reductase, the affinities varying within a range of two powers of ten (Ki = 0.3-32 microM). On variation of the substituents at C-3 of the acyl residue of the physiological substrate the affinity of both CoA-thioesters and CoA-thioethers increases in the sequence CH2, C(CH3)2, CH(CH3), C(OH)CH3, CH(OH).     

Carbon-fate maps for metabolic reactions

MOTIVATION: Stable isotope labeling of small-molecule metabolites (e.g. (13)C-labeling of glucose) is a powerful tool for characterizing pathways and reaction fluxes in a metabolic network. Analysis of isotope labeling patterns requires knowledge of the fates of individual atoms and moieties in reactions, which can be difficult to collect in a useful form when considering a large number of enzymatic reactions. RESULTS: We report carbon-fate maps for 4605 enzyme-catalyzed reactions documented in the KEGG database. Every fate map has been manually checked for consistency with known reaction mechanisms. A map includes a standardized structure-based identifier for each reactant (namely, an InChI string); indices for carbon atoms that are uniquely derived from the metabolite identifiers; structural data, including an identification of homotopic and prochiral carbon atoms; and a bijective map relating the corresponding carbon atoms in substrates and products. Fate maps are defined using the BioNetGen language (BNGL), a formal model-specification language, which allows a set of maps to be automatically translated into isotopomer mass-balance equations. AVAILABILITY: The carbon-fate maps and software for visualizing the maps are freely available (http://cellsignaling.lanl.gov/FateMaps/).     

Standard Gibbs energy of metabolic reactions: V. Enolase reaction

The glycolytic pathway is one of the most important pathways for living organisms, due to its role in energy production and as supplier of precursors for biosynthesis in living cells. This work focuses on determination of the standard Gibbs energy of reaction Δ^Rg′⁰ of the enolase reaction, the ninth reaction in the glycolysis pathway. Exact Δ^Rg′⁰ values are required to predict the thermodynamic feasibility of single metabolic reactions or even of metabolic reaction sequences under cytosolic conditions. So-called “apparent” standard data from literature are only valid at specific conditions. Nevertheless, such data are often used in pathway analyses, which might lead to misinterpretation of the results. In this work, equilibrium measurements were combined with activity coefficients in order to obtain new standard values Δ^Rg′⁰ for the enolase reaction that are independent of the cytosolic conditions. Reaction equilibria were measured at different initial substrate concentrations and temperatures of 298.15 K, 305.15 K and 310.15 K at pH 7. The activity coefficients were predicted using the equation of state electrolyte Perturbed-Chain Statistical Associating Fluid Theory (ePC-SAFT). The ePC-SAFT parameters were taken from literature or fitted to new experimentally determined osmotic coefficients and densities. At 298.15 K and pH 7, a Δ^Rg′⁰(298.15 K, pH 7) value of −2.8 ± 0.2 kJ mol⁻¹ was obtained. This value differs by up to 5 kJ mol⁻¹ from literature data. Reasons are the poorly defined “standard” conditions and partly undefined reaction conditions of literature works. Finally, using temperature-dependent equilibrium constants and the van ‘t Hoff equation, the standard enthalpy of reaction of Δ^Rh′⁰(298.15 K, pH 7) = 27 ± 10 kJ mol⁻¹ was determined, and a similar value was found by quantum-chemistry calculations.     

Temporal development of the barley leaf metabolic response to Pi limitation

The response of plants to P_i limitation involves interplay between root uptake of P_i, adjustment of resource allocation to different plant organs and increased metabolic P_i use efficiency. To identify potentially novel, early‐responding, metabolic hallmarks of P_i limitation in crop plants, we studied the metabolic response of barley leaves over the first 7 d of P_i stress, and the relationship of primary metabolites with leaf P_i levels and leaf biomass. The abundance of leaf P_i, Tyr and shikimate were significantly different between low Pi and control plants 1 h after transfer of the plants to low P_i. Combining these data with ¹⁵N metabolic labelling, we show that over the first 48 h of P_i limitation, metabolic flux through the N assimilation and aromatic amino acid pathways is increased. We propose that together with a shift in amino acid metabolism in the chloroplast a transient restoration of the energetic and redox state of the leaf is achieved. Correlation analysis of metabolite abundances revealed a central role for major amino acids in P_i stress, appearing to modulate partitioning of soluble sugars between amino acid and carboxylate synthesis, thereby limiting leaf biomass accumulation when external P_i is low.     

Engineering E. coli for triglyceride accumulation through native and heterologous metabolic reactions

Triglycerides, traditionally sourced from plant oils, are heavily used in both industrial and healthcare applications. Commercially significant products produced from triglycerides include biodiesel, lubricants, moisturizers, and oils for cooking and dietary supplements. The need to rely upon plant-based production, however, raises concerns of increasing demand and sustainability. The reliance on crop yields and a strong demand for triglycerides provides motivation to engineer production from a robust microbial platform. In this study, Escherichia coli was engineered to synthesize and accumulate triglycerides. Triglycerides were produced from cell wall phospholipid precursors through engineered expression of two enzymes, phosphatidic acid phosphatase (PAP) and diacylglycerol acyltransferase (DGAT). A liquid chromatography–mass spectrometry (LC–MS) method was developed to analyze the production of triglycerides by the engineered E. coli strains. This proof-of-concept study demonstrated a yield of 1.1 mg/L triglycerides (2 g/L dry cell weight) in lysogeny broth medium containing 5 g/L glucose at 8 h following induction of PAP and DGAT expression. LC–MS results also demonstrated that the intracellular triglyceride composition of E. coli was highly conserved. Triglycerides containing the fatty acid distributions 16:0/16:0/16:1, 16:0/16:0/18:1, and 18:1/16:0/16:1 were found in highest concentrations and represent ～70 % of triglycerides observed.     

High-throughput metabolic screening of microalgae genetic variation in response to nutrient limitation

Metabolomics - Microalgae produce metabolites that could be useful for applications in food, biofuel or fine chemical production. The identification and development of suitable strains require...     

Various metabolic reactions of formate in animal tissues

A new metabolic reaction of the aldolase condensation between formic acid and acetaldehyde proceeding with the formation of milk acid is detected in the liver of rats. Milk acid has been determined by chemical, enzymic and autoradiographic methods. Homogeneous preparations of the enzyme which catalyzes the mentioned reactions and is called lactate synthase are obtained in the crystalline form. The method for obtaining the lactate synthase from the rat liver is described as well as certain properties of the lactate synthase.     

Erythrocytic reactions and metabolic shifts in long-term experimental hypercatecholaminemia

The experiments on intact dogs have revealed that daily two-hour infusions of stressor norepinephrine doses are accompanied by the elevation in erythrocyte, hematocrit and hemoglobin levels, changes in circulating blood volume and signs of compensated metabolic acidosis. These adaptive responses become excessive in persistent hypercatecholaminemia and promote the development of stress-induced organic lesions.     

Effect of nitrogen limitation on foliar antioxidants in relationship to other metabolic characteristics

The long-term effect of limiting soil nitrogen (N) availability on foliar antioxidants, thermal energy dissipation, photosynthetic and respiratory electron transport, and carbohydrates was investigated in Spinacia oleracea L. Starch, sucrose, and glucose accumulated in leaves of N-limited spinach at predawn, consistent with a downregulation of chloroplast processes by whole-plant sink limitation in response to a limited supply of N-based macromolecules throughout the plant. On a leaf-area or dry-weight basis, levels of chlorophyll, carotenoid pools, photosynthetic electron transport capacity, as well as activities for the predominantly chloroplast-localized antioxidant enzymes ascorbate peroxidase (EC 1.11.1.11) and glutathione reductase (EC 1.6.4.2) were much lower in N-limited versus N-replete plants. When expressed on a chlorophyll basis, foliar levels of all of these parameters were similar in N-replete versus N-limited plants. However, on a total-protein basis, antioxidant enzyme activities were higher in N-limited plants. Nitrogen-limited spinach showed higher levels of thermal energy dissipation and of zeaxanthin and antheraxanthin at midday, as well as slightly higher ascorbate contents relative to chlorophyll. These results indicate that strong, long-term N limitation led not only to alterations in the balance between different processes but also to an overall downregulation of light collection, photosynthetic electron transport capacity, and chloroplast-based antioxidant enzymes. This is further supported by the finding that glucose-feeding of excised leaves led to strong concomitant decreases in photosynthetic electron transport capacity and ascorbate peroxidase activity. On a leaf-area basis, neither superoxide dismutase (EC 1.15.1.1) activity nor dark repiration rates showed a treatment effect. This indicates that overall mitochondrial electron transport activity does not decrease under long-term N limitation and is consistent with localization of an important fraction of foliar superoxide dismutase in mitochondria. Received: 19 March 1999 / Accepted: 13 April 1999