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Refolding and enzyme kinetic studies on the ferrochelatase of the cyanobacterium synechocystis sp. PCC 6803
Umeå University, Faculty of Science and Technology, Department of Chemistry.
Umeå University, Faculty of Science and Technology, Department of Chemistry. (Christiane Funk)
Umeå University, Faculty of Science and Technology, Department of Chemistry.
Umeå University, Faculty of Science and Technology, Department of Chemistry.
2013 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 2, e55569- p.Article in journal (Refereed) Published
Abstract [en]

Heme is a cofactor for proteins participating in many important cellular processes, including respiration, oxygen metabolism and oxygen binding. The key enzyme in the heme biosynthesis pathway is ferrochelatase (protohaem ferrolyase, EC 4.99.1.1), which catalyzes the insertion of ferrous iron into protoporphyrin IX. In higher plants, the ferrochelatase enzyme is localized not only in mitochondria, but also in chloroplasts. The plastidic type II ferrochelatase contains a C-terminal chlorophyll a/b (CAB) motif, a conserved hydrophobic stretch homologous to the CAB domain of plant light harvesting proteins and light-harvesting like proteins. This type II ferrochelatase, found in all photosynthetic organisms, is presumed to have evolved from the cyanobacterial ferrochelatase. Here we describe a detailed enzymological study on recombinant, refolded and functionally active type II ferrochelatase (FeCh) from the cyanobacterium Synechocystis sp. PCC 6803. A protocol was developed for the functional refolding and purification of the recombinant enzyme from inclusion bodies, without truncation products or soluble aggregates. The refolded FeCh is active in its monomeric form, however, addition of an N-terminal His6-tag has significant effects on its enzyme kinetics. Strikingly, removal of the C-terminal CAB-domain led to a greatly increased turnover number, kcat, compared to the full length protein. While pigments isolated from photosynthetic membranes decrease the activity of FeCh, direct pigment binding to the CAB domain of FeCh was not evident.

Place, publisher, year, edition, pages
2013. Vol. 8, no 2, e55569- p.
National Category
Chemical Sciences
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:umu:diva-59788DOI: 10.1371/journal.pone.0055569OAI: oai:DiVA.org:umu-59788DiVA: diva2:556618
Note

The authors are thankful to the Royal Swedish Academy (to C.F.) and the Kempe foundation (to P.S.) for granting their positions. The work was supported by the Swedish Energy Agency and Umea° University. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

 

Available from: 2012-09-25 Created: 2012-09-25 Last updated: 2017-12-07Bibliographically approved
In thesis
1. Functional studies on the Light-harvesting-Like (LiL) Proteins in Cyanobacteria and Cryptophytes
Open this publication in new window or tab >>Functional studies on the Light-harvesting-Like (LiL) Proteins in Cyanobacteria and Cryptophytes
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The light-harvesting like (LiL) proteins are a widely spread group of proteins within photosynthetic organisms. They are membrane proteins composed of one to four transmembrane helices and – in homology to the light-harvesting complexes of algae and higher plants – at least one of these transmembrane helices contains the chlorophyll a/b-binding (CAB) domain. Opposite to the light-harvesting antenna complexes, LiL proteins are stress induced and they have been shown to be involved in protection of the photosynthetic apparatus. The work presented in this thesis is focused on understanding the function of one-helical LiL proteins of the cryptophyte algae Guillardia theta and the cyanobacterium Synechocystis sp. PCC 6803. G. theta contains two genes encoding LiL proteins, one is localized in the plastid (hlipP), the other in the nucleomorph (HlipNm). Both genes are expressed in normal growth condition, but they are not induced by high light. Immunostaining indicated that HlipNm is translated, but not light-induced. These proteins therefore seem not to be involved in photoprotective mechanisms of G. theta. In the cyanobacterium Synechocystis sp. PCC 6803 four one-helical LiL proteins were identified, they are called Small CAB-like Proteins (SCPs); a fifth LiL (ScpA) is fused with the ferrochelatase (FC), an enzyme involved in the heme synthesis. Our analysis revealed that SCPs are involved in the de novo assembly/repair cycle of Photosystem II, stabilizing the chlorophyll pigments at their protein scaffold. The in vitro characterization of the recombinant FC showed that ScpA is involved in the product-release of the catalytic domain of the enzyme, thereby regulating substrate availability for chlorophyll- or heme- biosynthesis. Finally, using a transcriptomic and metabolomic approaches, I was able to show that deletion of all SCP genes has profound impact on the cell organization and metabolism. In SCP-depleted cells, production of reactive oxygen species (ROS) is increased, while the amount of Photosystem II per cell volume is decreased, causing a macronutrient-deficient phenotype. Therefore, SCPs are important for stress protection and help to maintain a metabolic equilibrium within the cell.

Place, publisher, year, edition, pages
Umeå: Umeå University, 2012. 67 p.
Keyword
Photosynthesis, cyanobacteria, Guillardia theta, photosystem II, chlorophyll-binding proteins, onehelix LiL proteins, photoprotection
National Category
Natural Sciences
Research subject
Biochemistry
Identifiers
urn:nbn:se:umu:diva-59801 (URN)978-91-7459-486-7 (ISBN)
Public defence
2012-10-26, KBC-huset, KB3A9, Umeå University, Umeå, 10:00 (English)
Opponent
Supervisors
Available from: 2012-10-05 Created: 2012-09-26 Last updated: 2012-09-26Bibliographically approved

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