Phosphoglycerate kinase acts as a futile cycle at high temperature

In (hyper)thermophilic organisms metabolic processes have to be adapted to function optimally at high temperature. We compared the gluconeogenic conversion of 3-phosphoglycerate via 1,3-bisphosphoglycerate to glyceraldehyde-3-phosphate at 30 C and at 70 C. At 30 C it was possible to produce 1,3-bisphosphoglycerate from 3-phosphoglycerate with phosphoglycerate kinase, but at 70 C, 1,3-bisphosphoglycerate was dephosphorylated rapidly to 3-phosphoglycerate, effectively turning the phosphoglycerate kinase into a futile cycle. When phosphoglycerate kinase was incubated together with glyceraldehyde 3-phosphate dehydrogenase it was possible to convert 3-phosphoglycerate to glyceraldehyde 3- phosphate, both at 30 C and at 70 C, however, at 70 C only low concentrations of product were observed due to thermal instability of glyceraldehyde 3-phosphate. Thus, thermolabile intermediates challenge central metabolic reactions and require special adaptation strategies for life at high temperature

The JWS online simulation database

Summary: JWS Online is a web-based platform for construction, simulation and exchange of models in standard formats. We have extended the platform with a database for curated simulation experiments that can be accessed directly via a URL, allowing one-click reproduction of published results. Users can modify the simulation experiments and export them in standard formats. The Simulation database thus lowers the bar on exploring computational models, helps users create valid simulation descriptions and improves the reproducibility of published simulation experiments. Availability and Implementation: The Simulation Database is available on line at https://jjj.bio.vu. nl/models/experiments/

Ph-dependence of the quaternary structure of the cyanide dihydratase from bacillus pumilus

Includes bibliographical references (leaves 81-86).Nitrilases are moderately ubiquitous nitrile/cyanide-degrading enzymes, found in both eukaryotes (animals, fungi, plants) and prokaryotes (archaea, bacteria) which catalyse the condensation and hydrolysis of a wide range of non-peptide nitrile substrates and are involved in nitrile-posttranslational modification. As Cyanide and related compounds are used extensively by humans in various industrial processes which, due to carelessness and inadequate waste-management systems, contribute significantly to the levels of toxic cyanide contamination in the environment nitrilases have been speculated to be useful for bioremediation amongst other things.Nitrile/cyanide hydrolysing enzymes have a broad range of substrates and they function via four known pathways. Nitrilase and cyanide dihydratase completely hydrolyse nitriles and HCN respectively to yield the corresponding acid and ammonia without going via an amide intermediate. Nitrile hydratase and cyanide hydratase perform a single hydrolysis producing the corresponding amide and formamide, respectively. The nitrilases are known to form extensive quaternary structures including dimers, spirals and rods/helices. Generally microbial nitrilases exist as homo-oligomers having a large molecular weight (>300 kDa). These enzymes are known to oligomerise under conditions of substrate activation (Rhodococcus rhodocrous) and pH change as is the case for the Cyanide dihydratase from Bacilluspumilus Cl (CynDpum) which exists as a terminating spiral of -16 subunits above pH 6 but forms a long helical fibre below -pH 6. In this project the Cyanide dihydratase from strain 8A3 of B. pumilus was analysed using electron microscopy at pH of 5.4,6 and 8. These data were reconstructed at pH 6 and pH 8 using the single particle reconstruction technique to resolutions of 29A and 31A respectively. It is shown that at pH 6 the enzyme consists of 20 subunits (10 dimers) and at pH 8 22 subunits (11 dimers). These models show that CynDpum exists as an oligomeric spiral that terminates by decreasing the helical radius and tilting the terminal subunits toward the helical axis. Below pH 5.4 CynDpum from strain 8A3 does not extend into a fibre as in Cl, this is explained to be due to the lack of 3 key histidine residues found on the C-terminal tail of CynDpum which point into the inner cavity of the spiral and become charged below pH 6 producing a repulsion preventing the termination of the spiral by narrowing of the helical radius and thus encouraging extension into the helical form

Determining enzyme kinetics for systems biology with nuclear magnetic resonance spectroscopy

CITATION: Eicher, J. J., Snoep, J. L. & Rohwer, J. M. 2012. Determining enzyme kinetics for systems biology with nuclear magnetic resonance spectroscopy. Metabolites, 2(4):818-843, doi:10.3390/metabo2040818.The original publication is available at http://www.mdpi.comEnzyme kinetics for systems biology should ideally yield information about the enzyme’s activity under in vivo conditions, including such reaction features as substrate cooperativity, reversibility and allostery, and be applicable to enzymatic reactions with multiple substrates. A large body of enzyme-kinetic data in the literature is based on the uni-substrate Michaelis–Menten equation, which makes unnatural assumptions about enzymatic reactions (e.g., irreversibility), and its application in systems biology models is therefore limited. To overcome this limitation, we have utilised NMR time-course data in a combined theoretical and experimental approach to parameterize the generic reversible Hill equation, which is capable of describing enzymatic reactions in terms of all the properties mentioned above and has fewer parameters than detailed mechanistic kinetic equations; these parameters are moreover defined operationally. Traditionally, enzyme kinetic data have been obtained from initial-rate studies, often using assays coupled to NAD(P)H-producing or NAD(P)H-consuming reactions. However, these assays are very labour-intensive, especially for detailed characterisation of multi-substrate reactions. We here present a cost-effective and relatively rapid method for obtaining enzyme-kinetic parameters from metabolite time-course data generated using NMR spectroscopy. The method requires fewer runs than traditional initial-rate studies and yields more information per experiment, as whole time-courses are analyzed and used for parameter fitting. Additionally, this approach allows real-time simultaneous quantification of all metabolites present in the assay system (including products and allosteric modifiers), which demonstrates the superiority of NMR over traditional spectrophotometric coupled enzyme assays. The methodology presented is applied to the elucidation of kinetic parameters for two coupled glycolytic enzymes from Escherichia coli (phosphoglucose isomerase and phosphofructokinase). 31P-NMR time-course data were collected by incubating cell extracts with substrates, products and modifiers at different initial concentrations. NMR kinetic data were subsequently processed using a custom software module written in the Python programming language, and globally fitted to appropriately modified Hill equations.http://www.mdpi.com/2218-1989/2/4/818Publisher's versio

Linking agribusiness and small-scale farmers in developing countries: Is there a new role for contract farming?

This article examines a new role for contract farming in developing countries in the light of the industrialisation of agriculture and the globalisation of world markets. A theoretical rationale for contracting in developing countries is developed on the basis of adopting new institutional economic theory for the purpose of matching governance forms to market failure problems and transaction characteristics. The history of contract farming is reviewed, together with the advantages and disadvantages to the various players, for the purpose of developing a list of key success factors, problems and some possible solutions.