Optimising photosynthesis is a significant goal for those researching ways to secure the human food supply on a planet that will continue to see the emergence of threats to agriculture from climate change. We’ve written a number of articles in this vein, predominantly on the effort to be able to engineer C4 photosynthesis into major crops that use the less efficient C3 photosynthesis (see here, here, here and here for a few examples).
In the research article “Plant RuBisCo assmbly in E. coli with five chloroplast chaperones including BSD2″ published in Science, researchers from the Max Planck Institute of Biochemistry sought to install a functional RuBisCo enzyme from our favourite model organism, Arabidopsis thaliana, into E. coli, the model bacteria with a short generation time.
The researchers explain that the purpose of attempting to install plant RuBisCo into the bacteria is to assist the effort seeking to improve the catalytic efficiency of the enzyme, allowing it to be studied closely in conjunction with genetic engineering to hasten the results of this line of research.
The genes that transcribe the subunits of RuBisCo have been known for some time and the arrangement of eight large subunits (RbcL) and eight small subunits (RbcS) is well characterised. But as with the ongoing quest to understand protein folding patterns, the process by which the holoenzyme comes to be in its final conformation, which supplementary proteins are required to assist the assembly of the enzyme and therefore which supplementary genes need to be expressed for the functional formation of the enzyme, are not well understood.
By attempting to build the enzyme from scratch, we learn more about the necessary genetic components required to build it, as these researchers found.
The researchers first attempted to express A. thaliana RuBisCo in E. coli by transferring the RbcL and RbcS genes along with two additional factors, RbcX and Raf1 (the functions of which were further elucidated later in the paper), but this attempt was unsuccessful.
Therefore, they attempted the feat again using three separate plasmids, one containing the RbcL and RcbS components, another containing chloroplast chaperonin proteins (C60αβ/C20), and a final plasmid containing four assembly chaperones (Raf1, Raf2, RbcX and BSD2). Using separate plasmids allowed the researchers to test which components were required to form functional enzymes. Expressing all genes resulted in the formation of proteins that migrated on polyacrylamide gel electrophoresis at the same position as AtRuBisCo and which had a similar affinity and carboxylation rate of CO2 as native RuBisCo. Plasmids that did not express either sets of chaperones failed to form proteins that migrated to the same position.
Accordingly, the RbcL and RbcS genes plus the genes encoding the various chaperones transferred into the E. coli appears to have resulted in the expression of functional RuBisCo.
Being able to functionally express RuBisCo from a higher order plant in E. coli is a significant step forward, but understanding the purpose and need for each of the chaperones remains outstanding. To probe a bit deeper into the requirements for functional RuBisCo enzymes, the researchers varied and substituted a number of the genes and monitored the outcome.
The first reported variation was to substitute the C60αβ/C20 chaperones with GroEL/GroES, endogenous chaperones from E. coli. The researchers found that even overexpressing the endogenous chaperones couldn’t match the level of functional expression of RuBisCo compared to when A. thaliana chaperones were used. Cpn60α and Cpn60β were both required for efficient expression of the enzyme, while expression of a plasmid without C20 still resulted in the formation of RuBisCo, albeit with a lower rate of expression. Overexpressing GroES in the absence of C20 resulted in the production of RuBisCo with similar efficiency as when C20 is coexpressed.
Figure 2 from article. Chloroplast charperonin requirements to form functional RuBisCo. Only when the RbcL, RbcS and folding chaperones were expressed with C60αβ/C20 (lane 2) or with C60αβ/GroES (lane 9) did RuBisCo successfully form.
In relation to the Raf1, Raf2, RbcX and BSD2 factors, the researchers deleted each of these in turn to test the effect on the final outcome of the enzyme to gain further insight into the function of each.
Deleting Raf1, Raf2 or BSD2 resulted in no RuBisCo production. Previous research has demonstrated that Raf1 works to assemble the RbcL subunits into RbcL8, whilst the function of Raf2 remains to be diagnosed.
RbcX deletion didn’t completely halt RuBisCo production but reduced assembly rates by 50% to 60%.
BSD2, what does it do?
The research delved much deeper into the function of BSD2, a gene that isn’t contained in all photosynthetic organisms and appears to have evolved subsequent to the endosymbiosis of the organism that led to chloroplasts, unlike the other three chaperones.
It was theorised in the paper that, given the possible evolutionary origins of the protein, that it may be related to the importation of the RbcS subunits into the chloroplast. When RbcS wasn’t expressed, a RbcL8/BSD2 complex formed. When RbcS was reintroduced, the RbcL8/BSD2 complex no longer formed; instead the RuBisCo RbcL8S8 enzyme formed. Omission of RbcS and Raf1 resulted in the lack of formation of the RbcL8/BSD2 protein, suggesting that Raf1 plays a role in the formation of this intermediary phase of the holoenzyme.
Given that it appears BSD2 plays a role late in the construction of the RbcL8S8 enzyme, the researchers tested whether altering the BSD2 and RbclS expression would have similar affects on the cyanobacterium Synechococcus elongatus. Without the expression of SeRbcS, SeRbcL and BSD2 formed a SeRbcL/BSD2 complex which, when SeRbcS was reinstroduced, resulted in fully formed SeRuBisCo, similar to what happened with the Arabidopsis based genes when expressed in E. coli. Therefore, this experiment helps to confirm the hypothses that BSD2 play a late but significant role in the final formation of RuBisCo.
Figure 6D from article. Model of protein folding sequence and required chaperone involvement to form functional RuBisCo.
Taking our knowledge of this process a step further, the structure of the BSD2 protein and how it may interact with RbcL to form a RbcL8/BSD2 complex were analysed.
BSD2 imaging showed that it forms a hairpin structure that centres around two zinc atoms. Imaged as part of the RbcL8/BSD2 complex, the crystallised structure shows that two BSD2 molecules are bound within antiparallel RbcL2 units, appearing to join the two RbcL subunits together. One of the zinc-containing regions of the BSD2 interacts with the C-terminal end of one RbcL unit while the other zinc-containing region interacts with the N-terminal of the adjacent RbcL subunit. It appears that the conformation of the RbcL subunits when interacting with BSD2 allows RbcS subunits to access and bind to the required sites on RbcL. Mutating the BSD2 at contact sites resulted in a loss of function of the protein.
This piece of research will hopefully assist to increase efficiency in RuBisCo research, research which aims to increase photosynthesis efficiency with resultant improvements in crop yields and food security. The research is somewhat Feynmann-esque, referring to the note scribbled on his chalkboard when he passed that:
“What I cannot create, I do not understand”. Richard Feynmann, February 1988
Although this research is useful in its application for further research, the attempt to recreate the enzyme from the scratch also helps us to further understand the function of those genes and moving us closer to the depth of knowledge Feynmann aspired to.
Great research resulting in a method that we expect to see in use and built upon in future.