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

Abstract

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.

Analysis of Loss-of-Function Mutants in Aspartate Kinase and Homoserine Dehydrogenase Genes Points to Complexity in the Regulation of Aspartate-Derived Amino Acid Contents

“…Our AK activity data showed that monofunctional AKs collectively contribute more to the overall AK activity than the dual-functional AK-HSDHs, which is consistent with the estimated values reported by Curien et al (2009). Curien et al (2009) proposed that AK1 is the primary contributor to overall AK activity, because among the three AKs, AK1 is least sensitive to Lys inhibition (Curien et al, 2007).…”

“…Moreover, Curien et al (2007) reported that AK1 is synergistically inhibited by Lys and SAM (Curien et al, 2007). Therefore, we measured the AK activity in near-physiological concentrations of Lys, Thr, Ala, and SAM, which are 70, 300, 400, and 20 mM, respectively (Curien et al, 2009). Under this condition, the mutation in AK1, AK2, AK3, AK-HSDH1, or AK-HSDH2 gene reduced the overall AK activity by 28%, 55%, 17%, 21%, or 27% to 35%, respectively (Fig.…”

“…Curien et al (2009) proposed that AK1 is the primary contributor to overall AK activity, because among the three AKs, AK1 is least sensitive to Lys inhibition (Curien et al, 2007). The estimation by Curien et al (2009) was based on the relative protein abundances in chloroplast stroma and the sensitivities of different AK and AK-HSDH isoforms to physiological concentrations of allosteric effectors.…”

“…Curien et al (2009) proposed that AK1 is the primary contributor to overall AK activity, because among the three AKs, AK1 is least sensitive to Lys inhibition (Curien et al, 2007). The estimation by Curien et al (2009) was based on the relative protein abundances in chloroplast stroma and the sensitivities of different AK and AK-HSDH isoforms to physiological concentrations of allosteric effectors. However, because of the cross reactions of antibodies between isoforms, Curien et al (2009) were only able to determine the total amount of AK isoforms and the total amount of AK-HSDH isoforms and unable to determine the relative abundances of each isoform.…”

Synthetic RNA devices to expedite the evolution of metabolite-producing microbes

“…To construct strains with different intracellular concentrations of lysine, we chose lysC, an aspartokinase isozyme, as a target to modulate the metabolic flux from oxaloacetate to lysine. In a previous study, a lysC knockout strain showed a lysine starvation response, reflecting the fact that the other two isozymes are regulated by methionine and threonine 22,23 . We found that the leader sequences of E. coli lysC positioned upstream of the green (Fig.…”