Characterization of an Arabidopsis thaliana cDNA encoding an S-adenosylmethionine-sensitive threonine synthase Threonine synthase from higher plants

AbstractAn Arabidopsis thaliana cDNA encoding an Sadenosylmethionine-sensitive threonine synthase (EC 4.2.99.2) has been isolated by functional complementation of an Escherichia coli mutant devoid of threonine synthase activity. Threonine synthase from A. thaliana was shown to be synthesized with a transit peptide. The recombinant protein is activated by Sadenosylmethionine in the same range as the plant threonine synthase and evidence is presented for an involvement of the N-terminal part of the mature enzyme in the sensitivity to Sadenosylmethionine

Evidence for Two Distinct Effector-Binding Sites in Threonine Deaminase by Site-Directed Mutagenesis, Kinetic, and Binding Experiments

A three-dimensional structure comparison between the dimeric regulatory serine-binding domain of Escherichia coli D-3-phosphoglycerate dehydrogenase [Schuller, D. J., Grant, G. A., and Banaszak, L. J. (1995) Nat. Struct. Biol. 2, 69-76] and the regulatory domain of E. coli threonine deaminase [Gallagher, D. T., Gilliland, G. L., Xiao, G., Zondlo, J., Fisher, K. E., Chinchilla, D. , and Eisenstein, E. (1998) Structure 6, 465-475] led us to make the hypothesis that threonine deaminase could have two binding sites per monomer. To test this hypothesis about the corresponding plant enzyme, site-directed mutagenesis was carried out on the recombinant Arabidopsis thaliana threonine deaminase. Kinetic and binding experiments demonstrated for the first time that each regulatory domain of the monomers of A. thaliana threonine deaminase possesses two different effector-binding sites constituted in part by Y449 and Y543. Our results demonstrate that Y449 belongs to a high-affinity binding site whose interaction with a first isoleucine induces conformational modifications yielding a conformer displaying a higher activity and with enhanced ability to bind a second isoleucine on a lower-affinity binding site containing Y543. Isoleucine interaction with this latter binding site is responsible for conformational modifications leading to final inhibition of the enzyme. Y449 interacts with both regulators, isoleucine and valine. However, interaction of valine with the high-affinity binding site induces different conformational modifications leading to reversal of isoleucine binding and reversal of inhibition

Biochemical and structural characterization of the Arabidopsis bifunctional enzyme dethiobiotin synthetase-diaminopelargonic acid aminotransferase: evidence for substrate channeling in biotin synthesis.

International audienceDiaminopelargonic acid aminotransferase (DAPA-AT) and dethiobiotin synthetase (DTBS) catalyze the antepenultimate and the penultimate steps, respectively, of biotin synthesis. Whereas DAPA-AT and DTBS are encoded by distinct genes in bacteria, in biotin-synthesizing eukaryotes (plants and most fungi), both activities are carried out by a single enzyme encoded by a bifunctional gene originating from the fusion of prokaryotic monofunctional ancestor genes. In few angiosperms, including Arabidopsis thaliana, this chimeric gene (named BIO3-BIO1) also produces a bicistronic transcript potentially encoding separate monofunctional proteins that can be produced following an alternative splicing mechanism. The functional significance of the occurrence of a bifunctional enzyme in biotin synthesis pathway in eukaryotes and the relative implication of each of the potential enzyme forms (bifunctional versus monofunctional) in the plant biotin pathway are unknown. In this study, we demonstrate that the BIO3-BIO1 fusion protein is the sole protein form produced by the BIO3-BIO1 locus in Arabidopsis. The enzyme catalyzes both DAPA-AT and DTBS reactions in vitro and is targeted to mitochondria in vivo. Our biochemical and kinetic characterizations of the pure recombinant enzyme show that in the course of the reaction, the DAPA intermediate is directly transferred from the DAPA-AT active site to the DTBS active site. Analysis of several structures of the enzyme crystallized in complex with and without its ligands reveals key structural elements involved for acquisition of bifunctionality and brings, together with mutagenesis experiments, additional evidences for substrate channeling

The Nexus Land-Use model, approach articulating biophysical potentials and economic dynamics to model competition for land-use

International audienceInteractions between food demand, biomass energy and forest preservation are driving both food prices and land-use changes. This study presents a new model called Nexus Land-Use which describes these interactions through a representation of agricultural intensification. The model combine biophysics and economics to calculate crop yields, food prices, and resulting pasture and cropland areas within 12 inter-connected regions. The representation of cropland production systems relies on a biomass production function derived from the crop yield response function to inputs and a spatially explicit distribution of potential crop yields prescribed from the Lund-Postdam-Jena global vegetation model for managed Land (LPJmL). The economic principles governing decisions about land-use and intensification are adapted from the Ricardian rent theory, assuming cost minimisation. The land-use modelling approach described in this paper makes it possible to explore interactions among different types of demand for biomass, including indirect effects on land-use change resulting from international trade. Yield variations induced by the possible expansion of croplands on less suitable lands are modelled by using regional land area distributions of potential yields, and a boundary between intensive and extensive production. Idealized scenarios exploring the impact of forest preservation policies or rising energy price on agricultural intensification are presented

A benzoxazine/substituted borazine composite coating: A new resin for improving the corrosion resistance of the pristine benzoxazine coating applied on aluminum

In this paper, laboratory synthesized Phenol-paraPhenyleneDiAmine (P-pPDA) benzoxazine containing different amounts of B-trimesityl-N-triphenylborazine was applied by spin coating on aluminum and thermally cured. The addition of the borazine derivative (borazine 1) does not appear to modify the curing characteristics of the P-pPDA matrix itself as shown by FTIR, DSC and DEA analyses; however, some interactions - chemical and/or physical (co-crystallization) – between P-pPDA and borazine 1 cannot be excluded. The microstructure of the composites is characterized by a two phase system consisting of a dispersion of nanosized (10–20 nm) clusters for the lowest borazine 1 concentration (0.5 wt%), evolving towards bigger (100–200 nm), agglomerated clusters for higher borazine 1 concentrations (3 wt%) and finally, continuous, dendritic structures within the P-pPDA matrix for the highest borazine 1 concentration (10 wt%). The benzoxazine composite coating containing 0.5 wt% trimesitylborazine derivative showed a largely increased and durable ability to protect the aluminum substrate. It is shown that a highly capacitive behavior and durable barrier properties can be obtained for P-pPDA coatings containing such a low amount of borazine derivative homogeneously dispersed in the benzoxazine matrix. For concentrations of 3 wt%, as agglomeration took place and dendrites appeared for the highest concentration of borazine derivative (10 wt%), the corrosion resistance decreased with time

Understanding the regulation of aspartate metabolism using a model based on measured kinetic parameters

The aspartate-derived amino-acid pathway from plants is well suited for analysing the function of the allosteric network of interactions in branched pathways. For this purpose, a detailed kinetic model of the system in the plant model Arabidopsis was constructed on the basis of in vitro kinetic measurements. The data, assembled into a mathematical model, reproduce in vivo measurements and also provide non-intuitive predictions. A crucial result is the identification of allosteric interactions whose function is not to couple demand and supply but to maintain a high independence between fluxes in competing pathways. In addition, the model shows that enzyme isoforms are not functionally redundant, because they contribute unequally to the flux and its regulation. Another result is the identification of the threonine concentration as the most sensitive variable in the system, suggesting a regulatory role for threonine at a higher level of integration

The accumulation of assembly intermediates of the mitochondrial complex I matrix arm is reduced by limiting glucose uptake in a neuronal-like model of MELAS syndrome

Ketogenic diet (KD) which combined carbohydrate restriction and the addition of ketone bodies has emerged as an alternative metabolic intervention used as an anticonvulsant therapy or to treat different types of neurological or mitochondrial disorders including MELAS syndrome. MELAS syndrome is a severe mitochondrial disease mainly due to the m.3243A > G mitochondrial DNA mutation. The broad success of KD is due to multiple beneficial mechanisms with distinct effects of very low carbohydrates and ketones. To evaluate the metabolic part of carbohydrate restriction, transmitochondrial neuronal-like cybrid cells carrying the m.3243A > G mutation, shown to be associated with a severe complex I deficiency was exposed during 3 weeks to glucose restriction. Mitochondrial enzyme defects were combined with an accumulation of complex I (CI) matrix intermediates in the untreated mutant cells, leading to a drastic reduction in CI driven respiration. The severe reduction of CI was also paralleled in post-mortem brain tissue of a MELAS patient carrying high mutant load. Importantly, lowering significantly glucose concentration in cell culture improved CI assembly with a significant reduction of matrix assembly intermediates and respiration capacities were restored in a sequential manner. In addition, OXPHOS protein expression and mitochondrial DNA copy number were significantly increased in mutant cells exposed to glucose restriction. The accumulation of CI matrix intermediates appeared as a hallmark of MELAS pathophysiology highlighting a critical pathophysiological mechanism involving CI disassembly, which can be alleviated by lowering glucose fuelling and the induction of mitochondrial biogenesis, emphasizing the usefulness of metabolic interventions in MELAS syndrome

Rev-erb beta regulates the expression of genes involved in lipid absorption in skeletal muscle cells - Evidence for cross-talk between orphan nuclear receptors and myokines

Rev-erbbeta is an orphan nuclear receptor that selectively blocks trans-activation mediated by the retinoic acid-related orphan receptor-alpha (RORalpha). RORalpha has been implicated in the regulation of high density lipoprotein cholesterol, lipid homeostasis, and inflammation. Rev-erbbeta and RORalpha are expressed in similar tissues, including skeletal muscle; however, the pathophysiological function of Rev-erbbeta has remained obscure. We hypothesize from the similar expression patterns, target genes, and overlapping cognate sequences of these nuclear receptors that Rev-erbbeta regulates lipid metabolism in skeletal muscle. This lean tissue accounts for > 30% of total body weight and 50% of energy expenditure. Moreover, this metabolically demanding tissue is a primary site of glucose disposal, fatty acid oxidation, and cholesterol efflux. Consequently, muscle has a significant role in insulin sensitivity, obesity, and the blood-lipid profile. We utilize ectopic expression in skeletal muscle cells to understand the regulatory role of Rev-erbbeta in this major mass peripheral tissue. Exogenous expression of a dominant negative version of mouse Rev-erbbeta decreases the expression of many genes involved in fatty acid/lipid absorption (including Cd36, and Fabp-3 and -4). Interestingly, we observed a robust induction (> 15-fold) in mRNA expression of interleukin-6, an exercise-induced myokine that regulates energy expenditure and inflammation. Furthermore, we observed the dramatic repression (> 20- fold) of myostatin mRNA, another myokine that is a negative regulator of muscle hypertrophy and hyperplasia that impacts on body fat accumulation. This study implicates Rev-erbbeta in the control of lipid and energy homoeostasis in skeletal muscle. In conclusion, we speculate that selective modulators of Rev-erbbeta may have therapeutic utility in the treatment of dyslipidemia and regulation of muscle growth