Regulation of Arabidopsis thaliana DAHP synthase by the chloroplast light -dark cycle

The shikimate pathway, found only in microorganisms and plants, is the common biosynthetic route leading to the production of the aromatic amino acids. The first committed step is the condensation of phosphoenolpyruvate and erythrose-4-phosphate, a reaction catalyzed by 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase. DAHP synthases from higher plants are found in the chloroplast. They contain an amino terminal sequence for plastid import and a carboxy terminal extension that is not present in their bacterial orthologs. The heterologous expression of the Arabidopsis thaliana genes DHS1, DHS2, and DHS3 in Escherichia coli yields three A. thaliana DAHP synthase isoforms that have a strict reducing agent dependence. The apparent dissociation constants for dithiothreitol (DTT) to the enzyme vary substantially among the isoforms. Half maximal enzyme activity for DHS2 is reached with about 3 μM DTT, whereas the corresponding values for DHS3 and DHS1 are about 13 and 43 μM, respectively. Chemical modification of DHS1 with iodoacetamide removes the dependence on reducing agents showing that enzymatic activity is regulated by the redox state of the enzyme through the formation of disulfide bridges. Purified A. thaliana DAHP synthase is activated by reduced thioredoxins (TRX) from spinach, Spirulina, and E. coli. Recombinant spinach TRX activates A. thaliana DAHP synthase with an apparent dissociation constant of about 0.2 μM. DHS1 is two fold more active in the presence of DTT and TRX than DTT alone. DHS1 has a strict Mn2+ dependence that is modulated by Mg2+. The enzyme is inactive in submicromolar concentrations of Mn2+ or mM concentrations of Mg2+ alone. When the metals are added together they have a synergistic effect, producing an active enzyme. This effect is strongly dependent on the pH. Under physiological conditions in the dark, DHS1 is inactive. During the light cycle when the pH rises, the synergism between Mn2+ and Mg 2+ produces an active enzyme. The dependence of the enzyme on light-generated reduced TRX and the pH modulated metal ion activation establishes convincingly that DAHP synthase is a key regulated enzyme of the chloroplast

Design and implementation of three incoherent feed-forward motif based biological concentration sensors

Synthetic biology is a useful tool to investigate the dynamics of small biological networks and to assess our capacity to predict their behavior from computational models. In this work we report the construction of three different synthetic networks in Escherichia coli based upon the incoherent feed-forward loop architecture. The steady state behavior of the networks was investigated experimentally and computationally under different mutational regimes in a population based assay. Our data shows that the three incoherent feed-forward networks, using three different macromolecular inhibitory elements, reproduce the behavior predicted from our computational model. We also demonstrate that specific biological motifs can be designed to generate similar behavior using different components. In addition we show how it is possible to tune the behavior of the networks in a predicable manner by applying suitable mutations to the inhibitory elements