Oligomerization and characteristics of phosphoenolpyruvate carboxylase in Synechococcus PCC 7002

Abstract

phosphoenolpyruvate carboxylase (pepc) is an essential enzyme in plants. A photosynthetic form is present both as dimer and tetramer in C4 and CAM metabolism. Additionally, non-photosynthetic PEPcs are also present. The single, non-photosynthetic PEPc of the unicellular cyanobacterium Synechococcus PCC 7002 (Synechococcus), involved in the tcA cycle, was examined. Using size exclusion chromatography (SEC) and small angle X-ray scattering (SAXS), we observed that PEPc in Synechococcus exists as both a dimer and a tetramer. This is the first demonstration of two different oligomerization states of a non-photosynthetic PEPc. High concentration of Mg 2+ , the substrate pep and a combination of low concentration of Mg 2+ and HCO 3 − induced the tetramer form of the carboxylase. Using SEC-SAXS analysis, we showed that the oligomerization state of the carboxylase is concentration dependent and that, among the available crystal structures of pepc, the scattering profile of PEPc of Synechococcus agrees best with the structure of pepc from Escherichia coli. in addition, the kinetics of the tetramer purified in presence of Mg 2+ using Sec, and of the mixed population purified in presence of Mg 2+ using a Strep-tagged column were examined. Moreover, the enzyme showed interesting allosteric regulation, being activated by succinate and inhibited by glutamine, and not affected by either malate, 2-oxoglutarate, aspartic acid or citric acid.

Synthetic Enzyme‐Catalyzed CO2 Fixation Reactions

“…Several decarboxylases employ divalent metal ions without requiring any organic cofactors. For example, 2,6‐dihydroxybenzoic acid decarboxylases (2,6‐DHBDs) [48] and 2, 3‐DHBDs [49] utilize Zn 2+ and Mg 2+ /Mn 2+ respectively to facilitate the carboxylation of dihydroxybenzenes, while the aliphatic carboxylase, phosphoenolpyruvate carboxylase (PEPC) [50,51] employs either Co 2+ , Mg 2+ , or Mn 2+. in its carboxylation process.…”

Beamline B21: high-throughput small-angle X-ray scattering at Diamond Light Source

“…SEC-SAXS is a powerful technique for biological SAXS (bioSAXS) data collection, as it allows purification of the sample during the SAXS measurement (David & Pé rez, 2009;Mathew et al, 2004). The inherent difficulties associated with bioSAXS, namely buffer matching and polydispersity, are mitigated by SEC-SAXS as it allows for the effective separation of monomeric, oligomeric and aggregated structures (Durall et al, 2020). SAXS measurements made across individual chromatographic peaks are averaged, producing a reliable measurement of a well defined particle [ Fig.…”