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

Allosteric monofunctional aspartate kinases from Arabidopsis.

International audiencePlant monofunctional aspartate kinase is unique among all aspartate kinases, showing synergistic inhibition by lysine and S-adenosyl-l-methionine (SAM). The Arabidopsis genome contains three genes for monofunctional aspartate kinases. We show that aspartate kinase 2 and aspartate kinase 3 are inhibited only by lysine, and that aspartate kinase 1 is inhibited in a synergistic manner by lysine and SAM. In the absence of SAM, aspartate kinase 1 displayed low apparent affinity for lysine compared to aspartate kinase 2 and aspartate kinase 3. In the presence of SAM, the apparent affinity of aspartate kinase 1 for lysine increased considerably, with K(0.5) values for lysine inhibition similar to those of aspartate kinase 2 and aspartate kinase 3. For all three enzymes, the inhibition resulted from an increase in the apparent K(m) values for the substrates ATP and aspartate. The mechanism of aspartate kinase 1 synergistic inhibition was characterized. Inhibition by lysine alone was fast, whereas synergistic inhibition by lysine plus SAM was very slow. SAM by itself had no effect on the enzyme activity, in accordance with equilibrium binding analyses indicating that SAM binding to aspartate kinase 1 requires prior binding of lysine. The three-dimensional structure of the aspartate kinase 1-Lys-SAM complex has been solved [Mas-Droux C, Curien G, Robert-Genthon M, Laurencin M, Ferrer JL & Dumas R (2006) Plant Cell18, 1681-1692]. Taken together, the data suggest that, upon binding to the inactive aspartate kinase 1-Lys complex, SAM promotes a slow conformational transition leading to formation of a stable aspartate kinase 1-Lys-SAM complex. The increase in aspartate kinase 1 apparent affinity for lysine in the presence of SAM thus results from the displacement of the unfavorable equilibrium between aspartate kinase 1 and aspartate kinase 1-Lys towards the inactive form

Baseplate Component TssK and Spatio-Temporal Assembly of T6SS in Pseudomonas aeruginosa

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Amino acid biosynthesis: New architectures in allosteric enzymes.

This review focuses on the allosteric controls in the Aspartate-derived and the branched-chain amino acid biosynthetic pathways examined both from kinetic and structural points of view. The objective is to show the differences that exist among the plant and microbial worlds concerning the allosteric regulation of these pathways and to unveil the structural bases of this diversity. Indeed, crystallographic structures of enzymes from these pathways have been determined in bacteria, fungi and plants, providing a wonderful opportunity to obtain insight into the acquisition and modulation of allosteric controls in the course of evolution. This will be examined using two enzymes, threonine synthase and the ACT domain containing enzyme aspartate kinase. In a last part, as many enzymes in these pathways display regulatory domains containing the conserved ACT module, the organization of ACT domains in this kind of allosteric enzymes will be reviewed, providing explanations for the variety of allosteric effectors and type of controls observed

A new mode of dimerization of allosteric enzymes with ACT domains revealed by the crystal structure of the aspartate kinase from Cyanobacteria.

International audienceAspartate kinases (AKs) can be divided in two subhomology divisions, AKalpha and AKbeta, depending on the presence of an extra sequence of about 60 amino acids, which is found only in the N-terminus of all AKalpha's. To date, the structures of AKalpha failed to provide a role for this additional N-terminal sequence. In this study, the structure of the AKbeta from the Cyanobacteria Synechocystis reveals that this supplementary sequence is linked to the dimerization mode of AKs. Its absence in AKbeta leads to the dimerization by the catalytic domain instead of involving the ACT domains [Pfam 01842; small regulatory domains initially found in AK, chorismate mutase and TyrA (prephenate dehydrogenase)] as observed in AKalpha. Thus, the structural analysis of the Synechocystis AKbeta revealed a dimer with a novel architecture. The four ACT domains of each monomer interact together and do not make any contact with those of the second monomer. The enzyme is inhibited synergistically by threonine and lysine with the binding of threonine first. The interaction between ACT1 and ACT4 or between ACT2 and ACT3 generates a threonine binding site and a lysine binding site at each interface, making a total of eight regulatory sites per dimer and allowing a fine-tuning of the AK activity by the end products, threonine and lysine

A Novel Organization of ACT Domains in Allosteric Enzymes Revealed by the Crystal Structure of Arabidopsis Aspartate Kinase

Asp kinase catalyzes the first step of the Asp-derived essential amino acid pathway in plants and microorganisms. Depending on the source organism, this enzyme contains up to four regulatory ACT domains and exhibits several isoforms under the control of a great variety of allosteric effectors. We report here the dimeric structure of a Lys and S-adenosylmethionine–sensitive Asp kinase isoform from Arabidopsis thaliana in complex with its two inhibitors. This work reveals the structure of an Asp kinase and an enzyme containing two ACT domains cocrystallized with its effectors. Only one ACT domain (ACT1) is implicated in effector binding. A loop involved in the binding of Lys and S-adenosylmethionine provides an explanation for the synergistic inhibition by these effectors. The presence of S-adenosylmethionine in the regulatory domain indicates that ACT domains are also able to bind nucleotides. The organization of ACT domains in the present structure is different from that observed in Thr deaminase and in the regulatory subunit of acetohydroxyacid synthase III

Species-specific recruitment of transcription factors dictates toxin expression

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cAMP and Vfr Control Exolysin Expression and Cytotoxicity of Pseudomonas aeruginosa Taxonomic Outliers

International audienceThe two-partner secretion system ExlBA, expressed by strains of Pseu-domonas aeruginosa belonging to the PA7 group, induces hemorrhage in lungs due to disruption of host cellular membranes. Here we demonstrate that the exlBA genes are controlled by a pathway consisting of cAMP and the virulence factor regulator (Vfr). Upon interaction with cAMP, Vfr binds directly to the exlBA promoter with high affinity (equilibrium binding constant [K eq ] of 2.5 nM). The exlB and exlA expression was diminished in the Vfr-negative mutant and upregulated with increased intracel-lular cAMP levels. The Vfr binding sequence in the exlBA promoter was mutated in situ, resulting in reduced cytotoxicity of the mutant, showing that Vfr is required for the exlBA expression during intoxication of epithelial cells. Vfr also regulates function of type 4 pili previously shown to facilitate ExlA activity on epithelial cells, which indicates that the cAMP/Vfr pathway coordinates these two factors needed for full cy-totoxicity. As in most P. aeruginosa strains, the adenylate cyclase CyaB is the main provider of cAMP for Vfr regulation during both in vitro growth and eukaryotic cell infection. We discovered that the absence of functional Vfr in the reference strain PA7 is caused by a frameshift in the gene and accounts for its reduced cytotoxicity, revealing the conservation of ExlBA control by the CyaB-cAMP/Vfr pathway in P. aeruginosa taxonomic outliers. IMPORTANCE The human opportunistic pathogen Pseudomonas aeruginosa provokes severe acute and chronic human infections associated with defined sets of vir-ulence factors. The main virulence determinant of P. aeruginosa taxonomic outliers is exolysin, a membrane-disrupting pore-forming toxin belonging to the two-partner secretion system ExlBA. In this work, we demonstrate that the conserved CyaB-cAMP/Vfr pathway controls cytotoxicity of outlier clinical strains through direct tran-scriptional activation of the exlBA operon. Therefore, despite the fact that the type III secretion system and exolysin are mutually exclusive in classical and outlier strains, respectively, these two major virulence determinants share similarities in their mechanisms of regulation

Unique Features of a Pseudomonas aeruginosa α2-Macroglobulin Homolog

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