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Oligomerization and characteristics of phosphoenolpyruvate carboxylase in Synechococcus PCC 7002
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Biology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Biology.ORCID iD: 0000-0001-9079-2774
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.ORCID iD: 0000-0001-7256-0275
2020 (English)In: Scientific Reports, E-ISSN 2045-2322, Vol. 10, article id 3607Article in journal (Refereed) Published
Abstract [en]

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 Mg2+, the substrate PEP and a combination of low concentration of Mg2+ and HCO3 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 Mg2+ using SEC, and of the mixed population purified in presence of Mg2+ 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.

Place, publisher, year, edition, pages
2020. Vol. 10, article id 3607
National Category
Biochemistry Molecular Biology
Identifiers
URN: urn:nbn:se:uu:diva-392227DOI: 10.1038/s41598-020-60249-2ISI: 000560076100001PubMedID: 32107404OAI: oai:DiVA.org:uu-392227DiVA, id: diva2:1347568
Funder
NordForsk, 82845
Note

Title in thesis list of papers: Phosphoenolpyruvate carboxylase in Synechococcus PCC 7002: Oligomerization, structure, and characteristics

Available from: 2019-09-01 Created: 2019-09-01 Last updated: 2025-02-20Bibliographically approved
In thesis
1. Increased Carbon Fixation for Chemical Production in Cyanobacteria
Open this publication in new window or tab >>Increased Carbon Fixation for Chemical Production in Cyanobacteria
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The combustion of fossil fuels has created many environmental problems, the major one, the greenhouse effect. Thus, we need solutions in order to replace fossil fuels and recycle the CO2 in the atmosphere. Renewable energies have created attention the last decades but electricity is the main energy form obtained. Photosynthetic organisms (including cyanobacteria) can be used as cell factories since they can convert solar energy to chemical energy. In addition, the requisites to grow them are few; light water, CO2 and inorganic nutrients. Cyanobacteria have been genetically engineered in order to produce numerous chemicals and fuels of human interest in direct processes. However, the amount of product obtained is still low. Increased carbon fixation in cyanobacteria results in higher production of carbon-based substances. This thesis focuses on the effects of overexpressing the native phosphoenolpyruvate carboxylase (PEPc) in the model cyanobacterium Synechocystis PCC 6803. PEPc is an essential enzyme and provides oxaloacetate, an intermediate of the tricarboxylic acid cycle (TCA cycle). The TCA cycle is involved in connecting the carbon and nitrogen metabolism in cyanobacteria. The strains were further engineered to produce ethylene and succinate, two examples of interests for the chemical and fuel industry. Strains with additional PEPc produced significantly more ethylene and succinate. Moreover, an in vitro characterization of PEPc from the cyanobacterium Synechococcus PCC 7002 was performed. The focus was on oligomerization state, kinetics and the structure of the carboxylase. This thesis demonstrates that increasing carbon fixation and discovering the bottlenecks in chemical production can lead to higher yields and gives us hope that cyanobacteria can be commercialized.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2019. p. 65
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1848
National Category
Engineering and Technology
Identifiers
urn:nbn:se:uu:diva-392234 (URN)978-91-513-0736-7 (ISBN)
Public defence
2019-10-18, Häggsalen, Ångströmlaboratoriet, Lägerhyddsvägen 1, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2019-09-25 Created: 2019-09-01 Last updated: 2019-10-15

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