Life’s building block carbon enters biology by its transformation into organic molecules from the gas carbon dioxide. The vast majority of this process is powered by light via photosynthesis and performed by the enzyme Rubisco. This enzyme thus represents the gateway for the flux of carbon into life. In contrast to its critical metabolic role, all encountered variants of Rubisco display relatively slow catalytic turnover rates, and inevitably catalyse a wasteful sidereaction: the oxygenation of ribulose 1,5-bisphosphate. It appears that over geological timescales, the complex and completely conserved reaction mechanism of the carboxylase reaction could not be tuned by molecular evolution, which in vivo only substitutes single amino acid residues at a time. The enzyme appears to be trapped on a local optimum in sequence space (Mueller-Cajar and Whitney, 2008).
To sustain life, organisms needed to compensate for these properties by adapting less immutable parts of their molecular machinery. The solution adopted by the majority of land plants resulted in dramatic overexpression of Rubisco, resulting in leaves where almost half of soluble protein is partitioned into Rubisco (Mueller-Cajar, 2018). Consequently the enzyme is believed to be the most abundant protein on Earth. In addition to overexpression, a myriad of fascinating Rubisco compensation mechanisms has evolved, frequently from different molecular starting points. The careful molecular characterization and utilisation of Rubisco compensation mechanisms occupies the majority of the group’s time. In this context we initially focussed our attention on multiple novel molecular chaperones, that monitor the state of Rubisco active sites, and remove trapped inhibitors (Mueller-Cajar, 2017). Three classes of “Rubisco activase” have now been described, all belonging to the AAA+ family of molecular chaperones, but utilising highly dissimilar mechanisms to achieve their objective. For example the Rubisco activase (Rca) from red algae transiently threads the C-terminus of the Rubisco large subunit through the central pore of a disc-shaped ring, whereas the CbbQO Rca found in proteobacteria appears to function by transmitting force from the AAA+ ring through the CbbO adaptor protein.
Mechanistic model of red-type Rubisco activation (e.g. in red algae) by the CbbX-type Rubisco activase
A very wide-spread strategy towards saturating Rubisco active sites with its substrate CO2 and at the same time outcompete the competing substrate oxygen are the carbon concentrating mechanisms (CCMs). In most eukaryotic microalgae this is achieved by compartmentalisation of Rubisco in a chloroplast-localised membraneless organelle (MLO) known as the pyrenoid. We were able to demonstrate that only two proteins, the intrinsically disordered repeat protein EPYC1 and Rubisco were sufficient to recapitulate the formation of dense Rubisco droplets in vitro. This finding provides an in vitro system that will allow the careful mechanistic dissection of the pyrenoid-based CCM (Wunder et al. 2019). Similar to Rubisco activases, pyrenoids are highly diverse, and by examining different algae we are bound to find many molecular variations to the CCM solution.
Reconstitution of the EPYC1-Rubisco liquid liquid phase separation (Wunder et al. 2018)
Many colleagues worldwide are excited about the potential of employing the many Rubisco compensation strategies towards enhancing agriculture, by engineering crop plants. We aspire to provide information that will facilitate this goal. In addition the current increase of the greenhouse gas CO2 in our atmosphere suggests that humanity should prioritise learning as much as it can as soon as possible concerning the mechanisms the biological world uses to extract the gas.