eQuilibrator--the biochemical thermodynamics calculator

Flamholz, Avi I.; Noor, Elad; Bar‐Even, Arren; Milo, Ron

doi:10.1093/nar/gkr874

Cited by 527 publications

(569 citation statements)

References 27 publications

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“…First, incubation of BAL with 13 C-FALD led to new peaks in the NMR spectra. The chemical shifts and J-coupling correspond to the expected two carbon [glycolaldehyde (GALD), first carbon-carbon coupling of two FALDs] and the three-carbon (DHA) 13 C products of the formose reaction (SI Appendix, Fig. S2).…”

Section: Resultsmentioning

confidence: 89%

“…Formyl-CoA is not produced or hydrolyzed in any known primary metabolic pathway of E. coli (11). In addition, under standard E. coli cytoplasmic conditions, the ATP-dependent formation of formyl-CoA can have a Δ r G′°as low as −4 kcal/mol (12,13). Because E. coli has an ACS enzyme, we cloned the gene from the genomic DNA, and the corresponding enzyme (ecACS) was expressed and purified as described in Materials and Methods (14,15).…”

Section: Resultsmentioning

confidence: 99%

“…1D): (i) generation of reducing equivalents by FDH, (ii) carbon activation and reduction by ecACS and lmACDH, (iii) carbon-carbon bond formation by FLS, and (iv) assimilation into a central metabolite by phosphorylation of DHA using DHA kinase (DHAK). To explore the ability for this pathway to function in vitro, we incubated 13 C-formate with purified FDH, ecACS, lmACDH, FLS, and DHAK at room temperature and followed the appearance of 13 C-labeled DHAP using LC-MS/MS. In the presence of all of the enzymes, substrates, and cofactors, 13 C-DHAP was clearly observed and increased linearly over time (Fig.…”

Section: Resultsmentioning

confidence: 99%

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Computational protein design enables a novel one-carbon assimilation pathway

Siegel¹,

Smith²,

Poust

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

323

255

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We describe a computationally designed enzyme, formolase (FLS), which catalyzes the carboligation of three one-carbon formaldehyde molecules into one three-carbon dihydroxyacetone molecule. The existence of FLS enables the design of a new carbon fixation pathway, the formolase pathway, consisting of a small number of thermodynamically favorable chemical transformations that convert formate into a three-carbon sugar in central metabolism. The formolase pathway is predicted to use carbon more efficiently and with less backward flux than any naturally occurring one-carbon assimilation pathway. When supplemented with enzymes carrying out the other steps in the pathway, FLS converts formate into dihydroxyacetone phosphate and other central metabolites in vitro. These results demonstrate how modern protein engineering and design tools can facilitate the construction of a completely new biosynthetic pathway.computational protein design | pathway engineering | carbon fixation N ovel strategies are needed to address current challenges in energy storage and carbon sequestration. One approach is to engineer biological systems to convert one-carbon compounds into multicarbon molecules such as fuels and other high value chemicals. Many synthetic pathways to produce value-added chemicals from common feedstocks, such as glucose, have been constructed in organisms that lack one-carbon anabolic pathways, such as Escherichia coli or Saccharomyces cerevisiae (1-3); however, despite considerable effort, it has been difficult to introduce heterologous one-carbon fixing pathways into these organisms (4). Likely problems include the inherent complexity, environmental sensitivity, inefficiency, or unfavorable chemical driving force of naturally occurring one-carbon metabolic pathways (5).An optimal pathway for one-carbon utilization in common synthetic biology platforms would be (i) composed of a minimal number of enzymes, (ii) linear and disconnected from other metabolic pathways, (iii) thermodynamically favorable with a significant driving force at most or all steps, and (iv) capable of functioning in a robust manner under both aerobic and anaerobic conditions (5). A pathway with these properties could enable the assimilation of one-carbon molecules as the sole carbon source for the production of fuels and chemicals. Although no such pathway is known in nature, the established electrochemical reduction of carbon dioxide to formate under ambient temperatures and pressures in neutral aqueous solutions provides an attractive starting point for a onecarbon fixation pathway (5-8).We describe the computational design of an enzyme that catalyzes the carboligation of three one-carbon molecules into a single three-carbon molecule. This enzyme enables the construction of a new pathway, the formolase pathway, in which formate is converted into the central metabolite dihydroxyacetone phosphate (DHAP; Fig. 1). The use of computational protein design to reengineer catalytic activities opens up the pathway design space beyond that available based o...

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Section: Resultsmentioning

confidence: 89%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Computational protein design enables a novel one-carbon assimilation pathway

Siegel¹,

Smith²,

Poust

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

323

255

View full text Add to dashboard Cite

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“…eQuilibrator is effective online systems biology search tool which connects a comprehensive and accurate database of thermodynamic properties of biochemical compounds and enzymatic reactions. It empowers easy Gibb's energies (in Kcal/mol units) calculation of compounds and reactions considering arbitrary pH, ionic strength and metabolite concentrations in an online interactive interface (Flamholz et al, 2012). Phylogenetic analysis has been carried out using Phylogeny.fr platform (Dereeper et al, 2008) web server (i.e.…”

Section: Methodsmentioning

confidence: 99%

Systems Biology paves Pathway and Potential Enzymes Predictions towards Anticancer Drug Methyl Jasmonate Biosynthesis

2017

J App Pharm Sci

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Methyl Jasmonate (MJ) is a potential anticancer drug along with other therapeutic importance. MJ generation is predominantly dependent on plant based route which directly or indirectly interferes with ecosystem and environmental concerns. However, existing microbial platforms are not yet reliable enough to fulfil industrial prospects upon MJ large scale production due to lower productivity, titer and molar yield. To this end, the major objective of this study is to identify an efficient putative metabolic pathway and its corresponding enzyme components following in silico systems biology tools. Main aim of this study is to establish a platform or hypothesis following systems biology which has a great potential to design efficient pathway with corresponding enzymes towards improving MJ biosynthesis in near future. This computational approach helps to design two most promising intermediate metabolic routes of stearic acid to oleic acid biocatalytic conversions considering thermodynamic constraint towards MJ biosynthesis. Furthermore, this in silico combinatorial methodology predicts most energetically favorable downstream bioconversion of oleic acid for MJ biosynthetic circuit design along with novel enzymes identifications. Moreover, the future plan will be to functionalize and validate the entire predicted metabolic pathway through in vivo experimentation in suitable chassis microorganisms for ameliorating MJ biosynthesis.

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“…For all unmeasured metabolites in this model we used conservative default concentration ranges between

c_{k}^{truemin} = 10^{- 7} m

and

c_{k}^{truemax} = 1 m

. Δ f G 0 values were obtained from the online version of equilibrator 44. For two metabolites (ubiquinol‐8 and biomass) no Δ f G values were available.…”

Section: Methodsmentioning

confidence: 99%

Which sets of elementary flux modes form thermodynamically feasible flux distributions?

et al. 2016

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Elementary flux modes (EFMs) are non‐decomposable steady‐state fluxes through metabolic networks. Every possible flux through a network can be described as a superposition of EFMs. The definition of EFMs is based on the stoichiometry of the network, and it has been shown previously that not all EFMs are thermodynamically feasible. These infeasible EFMs cannot contribute to a biologically meaningful flux distribution. In this work, we show that a set of thermodynamically feasible EFMs need not be thermodynamically consistent. We use first principles of thermodynamics to define the feasibility of a flux distribution and present a method to compute the largest thermodynamically consistent sets (LTCSs) of EFMs. An LTCS contains the maximum number of EFMs that can be combined to form a thermodynamically feasible flux distribution. As a case study we analyze all LTCSs found in Escherichia coli when grown on glucose and show that only one LTCS shows the required phenotypical properties. Using our method, we find that in our E. coli model < 10% of all EFMs are thermodynamically relevant.

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eQuilibrator--the biochemical thermodynamics calculator

Cited by 527 publications

References 27 publications

Computational protein design enables a novel one-carbon assimilation pathway

Computational protein design enables a novel one-carbon assimilation pathway

Systems Biology paves Pathway and Potential Enzymes Predictions towards Anticancer Drug Methyl Jasmonate Biosynthesis

Which sets of elementary flux modes form thermodynamically feasible flux distributions?

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