Two phosphoenolpyruvate carboxykinases with differing biochemical properties in Chlamydomonas reinhardtii

Abstract

Phosphoenolpyruvate carboxykinase (PEPCK) catalyses the reversible reaction of decarboxylation and phosphorylation of oxaloacetate (OAA) to generate phosphoenolpyruvate (PEP) and CO2 playing mainly a gluconeogenic role in green algae. We found two PEPCK isoforms in Chlamydomonas reinhardtii and we cloned, purified and characterised both enzymes. ChlrePEPCK1 is more active as decarboxylase than ChlrePEPCK2. ChlrePEPCK1 is hexameric and its activity is affected by citrate, phenylalanine and malate, while ChlrePEPCK2 is monomeric and it is regulated by citrate, phenylalanine and glutamine. We postulate that the two PEPCK isoforms found originate from alternative splicing of the gene or regulated proteolysis of the enzyme. The presence of these two isoforms would be part of a mechanism to finely regulate the biological activity of PEPCKs.

The activation ofChlamydomonas reinhardtiialpha amylase 2 by glutamine requires its N-terminal ACT domain

“…5A) is close to the EC50 of Gln Chellamuthu et al (2014) determined for the activation of NAGK by the PII protein (2.4  0.8 mM), suggesting that both regulatory effects might be operative under similar physiological conditions. Rather recently, it was shown that Chlamydomonas phosphoenolpyruvate carboxykinase isoform 2 is inhibited by Gln (Torresi et al, 2023) is shown as gray cartoon, except for the ACT domains, in which consecutive -helices and -strands were colored in shades of green. The AMA2 dimer model and the HvAMY2 structure (purple cartoon)…”

“…5A) is close to the EC 50 o f Gln Chellamuthu et al (2014) determined for the activation of NAGK by the P II p rotein (2.4 ± 0.8 mM), suggesting that both regulatory effects might be operative under similar physiological conditions. Rather recently, it was shown that Chlamydomonas phosphoenolpyruvate carboxykinase isoform 2 is inhibited by Gln (Torresi et al, 2023), representing an additional example of a direct effect of Gln. It appears that the alga employs Gln as a signaling molecule to coordinate N- and C metabolism directly at the enzymatic level in diverse contexts, and that AMA2 might be an additional example.…”

Antagonistic effects of Talaromyces muroii TM28 against Fusarium crown rot of wheat caused by Fusarium pseudograminearum

“…Consequently, oxaloacetic acid and acetyl-CoA cannot be converted into citric acid in time and enter the TCA cycle ( Llamas-Ramirez et al, 2020 ; Tovilla-Coutino et al, 2020 ; Jezewski et al, 2021 ), ultimately affecting Fp metabolism and physiological balance. Phosphoenolpyruvate, as a crucial intermediate linking glycolysis and the TCA cycle, contributes to the amount of ATP required for cellular energy metabolism ( Torresi et al, 2023 ). Oxaloacetate and pyruvate are catalyzed by PEPCK and PEPs to generate phosphoenolpyruvate, TM28 downregulates the expression of PEPCK, and PEPs further inhibit TCA cycling and ATP energy production in Fp .…”

The activation of Chlamydomonas reinhardtii alpha amylase 2 by glutamine requires its N‐terminal aspartate kinase–chorismate mutase–tyrA (ACT) domain

“…Notably, the EC 50 value of 2.1 ± .7 mM of Gln we determined for AMA2 activation (Figure 5a ) is close to the EC 50 of Gln Chellamuthu et al ( 2014 ) determined for the activation of N ‐acetyl‐L‐glutamate kinase by the P II protein (2.4 ± .8 mM), suggesting that both regulatory effects might be operative under similar physiological conditions. Rather recently, it was shown that Chlamydomonas phosphoenolpyruvate carboxykinase isoform 2 is inhibited by Gln (Torresi et al, 2023 ), representing an additional example of a direct effect of Gln. It appears that the alga employs Gln as a signaling molecule to coordinate N and C metabolism directly at the enzymatic level in diverse contexts and that AMA2 might be an additional example.…”