RESUMO
How organisms integrate metabolism with the external environment is a central question in biology. Here, we describe a novel regulatory small molecule, a proteogenic dipeptide Tyr-Asp, which improves plant tolerance to oxidative stress by directly interfering with glucose metabolism. Specifically, Tyr-Asp inhibits the activity of a key glycolytic enzyme, glyceraldehyde 3-phosphate dehydrogenase (GAPC), and redirects glucose toward pentose phosphate pathway (PPP) and NADPH production. In line with the metabolic data, Tyr-Asp supplementation improved the growth performance of both Arabidopsis and tobacco seedlings subjected to oxidative stress conditions. Moreover, inhibition of Arabidopsis phosphoenolpyruvate carboxykinase (PEPCK) activity by a group of branched-chain amino acid-containing dipeptides, but not by Tyr-Asp, points to a multisite regulation of glycolytic/gluconeogenic pathway by dipeptides. In summary, our results open the intriguing possibility that proteogenic dipeptides act as evolutionarily conserved small-molecule regulators at the nexus of stress, protein degradation, and metabolism.
Assuntos
Arabidopsis/efeitos dos fármacos , Dipeptídeos/farmacologia , Gliceraldeído-3-Fosfato Desidrogenases/antagonistas & inibidores , Nicotiana/efeitos dos fármacos , Proteínas de Plantas/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Simulação por Computador , Dipeptídeos/química , Dipeptídeos/metabolismo , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/química , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/metabolismo , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , NADP/metabolismo , Oxirredução , Estresse Oxidativo/efeitos dos fármacos , Via de Pentose Fosfato/efeitos dos fármacos , Fosfoenolpiruvato Carboxiquinase (ATP)/metabolismo , Proteínas de Plantas/antagonistas & inibidores , Plântula/efeitos dos fármacos , Plântula/metabolismo , Nicotiana/metabolismoRESUMO
ATP-dependent phosphoenolpyruvate carboxykinases (PEPCKs, EC 4.1.1.49) from C4 and CAM plants have been widely studied due to their crucial role in photosynthetic CO2 fixation. However, our knowledge on the structural, kinetic and regulatory properties of the enzymes from C3 species is still limited. In this work, we report the recombinant production and biochemical characterization of two PEPCKs identified in Arabidopsis thaliana: AthPEPCK1 and AthPEPCK2. We found that both enzymes exhibited high affinity for oxaloacetate and ATP, reinforcing their role as decarboxylases. We employed a high-throughput screening for putative allosteric regulators using differential scanning fluorometry and confirmed their effect on enzyme activity by performing enzyme kinetics. AthPEPCK1 and AthPEPCK2 are allosterically modulated by key intermediates of plant metabolism, namely succinate, fumarate, citrate and α-ketoglutarate. Interestingly, malate activated and glucose 6-phosphate inhibited AthPEPCK1 but had no effect on AthPEPCK2. Overall, our results demonstrate that the enzymes involved in the critical metabolic node constituted by phosphoenolpyruvate are targets of fine allosteric regulation.