Mass spectrometry analysis of the photosystem II assembly factor Psb27 revealed variations in its lipid modification.

The assembly of large, multi-cofactor membrane protein complexes like photosystem II (PSII) requires a high level of coordination. The process is facilitated by a large network of auxiliary proteins that bind transiently to unassembled subunits, preassembled modules or intermediate states of PSII, which are comprised of a subset of subunits. However, analysis of these immature, partially assembled PSII complexes is hampered by their low abundance and intrinsic instability. In this study, PSII was purified from the thermophilic cyanobacterium Thermosynechococcus elongatus via Twin-Strep-tagged CP43 and further separated by ion exchange chromatography into mature and immature complexes. Mass spectrometry analysis of the immature Psb27-PSII intermediate revealed six different Psb27 proteoforms with distinct lipid modifications. The maturation and functional role of thylakoid localized lipoproteins are discussed.

Structural insights into photosystem II assembly.

Biogenesis of photosystem II (PSII), nature's water-splitting catalyst, is assisted by auxiliary proteins that form transient complexes with PSII components to facilitate stepwise assembly events. Using cryo-electron microscopy, we solved the structure of such a PSII assembly intermediate from Thermosynechococcus elongatus at 2.94 Å resolution. It contains three assembly factors (Psb27, Psb28 and Psb34) and provides detailed insights into their molecular function. Binding of Psb28 induces large conformational changes at the PSII acceptor side, which distort the binding pocket of the mobile quinone (QB) and replace the bicarbonate ligand of non-haem iron with glutamate, a structural motif found in reaction centres of non-oxygenic photosynthetic bacteria. These results reveal mechanisms that protect PSII from damage during biogenesis until water splitting is activated. Our structure further demonstrates how the PSII active site is prepared for the incorporation of the Mn4CaO5 cluster, which performs the unique water-splitting reaction.

Analysis of photosystem II biogenesis in cyanobacteria.

Photosystem II (PSII), a large multisubunit membrane protein complex found in the thylakoid membranes of cyanobacteria, algae and plants, catalyzes light-driven oxygen evolution from water and reduction of plastoquinone. Biogenesis of PSII requires coordinated assembly of at least 20 protein subunits, as well as incorporation of various organic and inorganic cofactors. The stepwise assembly process is facilitated by numerous protein factors that have been identified in recent years. Further analysis of this process requires the development or refinement of specific methods for the identification of novel assembly factors and, in particular, elucidation of the unique role of each. Here we summarize current knowledge of PSII biogenesis in cyanobacteria, focusing primarily on the impact of methodological advances and innovations. This article is part of a Special Issue entitled Organization and dynamics of bioenergetic systems in bacteria, edited by Conrad Mullineaux.

Photosynthetic production of enantioselective biocatalysts.

BACKGROUND: Global resource depletion poses a dramatic threat to our society and creates a strong demand for alternative resources that do not compete with the production of food. Meeting this challenge requires a thorough rethinking of all steps of the value chain regarding their sustainability resource demand and the possibility to substitute current, petrol-based supply-chains with renewable resources. This regards also the production of catalysts for chemical synthesis. Phototrophic microorganisms have attracted considerable attention as a biomanufacturing platform for the sustainable production of chemicals and biofuels. They allow the direct utilization of carbon dioxide and do not compete with food production. Photosynthetic enzyme production of catalysts would be a sustainable supply of these important components of the biotechnological and chemical industries. This paper focuses on the usefulness of recombinant cyanobacteria for the photosynthetic expression of enantioselective catalysts. As a proof of concept, we used the cyanobacterium Synechocystis sp. PCC 6803 for the heterologous expression of two highly enantioselective enzymes. RESULTS: We investigated the expression yield and the usefulness of cyanobacterial cell extracts for conducting stereoselective reactions. The cyanobacterial enzyme expression achieved protein yields of 3% of total soluble protein (%TSP) while the expression in E. coli yielded 6-8% TSP. Cell-free extracts from a recombinant strain expressing the recombinant esterase ST0071 from the thermophilic organism Sulfolobus tokodai ST0071 and arylmalonate decarboxylase from Bordetella bronchiseptica showed excellent enantioselectivity (>99% ee) and yield (>91%) in the desymmetrisation of prochiral malonates. CONCLUSIONS: We were able to present the proof-of-concept of photoautotrophic enzyme expression as a viable alternative to heterotrophic expression hosts. Our results show that the introduction of foreign genes is straightforward. Cell components from Synechocystis did not interfere with the stereoselective transformations, underlining the usability of photoautotrophic organisms for the production of enzymes. Given the considerable commercial value of recombinant biocatalysts, cyanobacterial enzyme expression has thus the potential to complement existing approaches to use phototrophic organisms for the production of chemicals and biofuels.

Cloning, expression, crystallization and preliminary X-ray studies of a superfolder GFP fusion of cyanobacterial Psb32.

A fusion of Psb32 from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 (TePsb32) with superfolder GFP was created for enhanced solubility and improved detection and purification. The fusion protein readily formed large hexagonal crystals belonging to space group P6122. A full data set extending to 2.3 Šresolution was collected at the Swiss Light Source. The phase problem could be solved by using only the sfGFP fusion partner or by using GFP and AtTLP18.3 from Arabidopsis thaliana as search models. Based on this expression construct, a versatile library of 24 vectors combining four different superfolder GFP variants and three affinity tags was generated to facilitate expression and screening of fluorescent fusion proteins.

Cloning, expression, crystallization and preliminary X-ray studies of the ferredoxin-NAD(P)+ reductase from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1.

Ferredoxin-NADP(+) reductase (FNR) is a flavoenzyme that catalyses the reduction of NADP(+) in the final step of the photosynthetic electron-transport chain. FNR from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 (TeFNR) contains an additional 9 kDa domain at its N-terminus relative to chloroplastic FNRs and is more thermostable than those from mesophilic cyanobacteria. With the aim of understanding the structural basis of the thermostability of TeFNR and assigning a structural role to the small additional domain, the gene encoding TeFNR with and without an additional domain was engineered for heterologous expression and the recombinant proteins were purified and crystallized. Crystals of TeFNR without the additional domain belonged to space group P2(1), with unit-cell parameters a = 55.05, b = 71.66, c = 89.73 Å, α = 90, ß = 98.21, γ = 90°.