Characterization of the heterooligomeric red-type rubisco activase from red algae

Abstract

The photosynthetic CO 2 -fixing enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (rubisco) is inhibited by nonproductive binding of its substrate ribulose-1,5-bisphosphate (RuBP) and other sugar phosphates. Reactivation requires ATP-hydrolysis-powered remodeling of the inhibited complexes by diverse molecular chaperones known as rubisco activases (Rcas). Eukaryotic phytoplankton of the red plastid lineage contain so-called red-type rubiscos, some of which have been shown to possess superior kinetic properties to green-type rubiscos found in higher plants. These organisms are known to encode multiple homologs of CbbX, the α-proteobacterial red-type activase. Here we show that the gene products of two cbbX genes encoded by the nuclear and plastid genomes of the red algae Cyanidioschyzon merolae are nonfunctional in isolation, but together form a thermostable heterooligomeric Rca that can use both α-proteobacterial and red algal-inhibited rubisco complexes as a substrate. The mechanism of rubisco activation appears conserved between the bacterial and the algal systems and involves threading of the rubisco large subunit C terminus. Whereas binding of the allosteric regulator RuBP induces oligomeric transitions to the bacterial activase, it merely enhances the kinetics of ATP hydrolysis in the algal enzyme. Mutational analysis of nuclear and plastid isoforms demonstrates strong coordination between the subunits and implicates the nuclearencoded subunit as being functionally dominant. The plastid-encoded subunit may be catalytically inert. Efforts to enhance crop photosynthesis by transplanting red algal rubiscos with enhanced kinetics will need to take into account the requirement for a compatible Rca.rubisco | activase | photosynthesis | AAA + proteins | red algae

In Vitro Characterization of Thermostable CAM Rubisco Activase Reveals a Rubisco Interacting Surface Loop

“…To our knowledge, agave Rca presents as the most thermostable green-type Rca in the absence of stabilizing ligands. Interestingly, the red-type Rca from the thermophilic red algae Cyanidioschyzon merolae tolerated incubation at 60°C (Loganathan et al, 2016); however, it is not compatible with plant Rubisco.…”

“…Rca activity was measured using an adaptation (Tsai et al, 2015) of the spectrophotometric Rubisco activase assay (Lan and Mott, 1991) and quantified as described (Loganathan et al, 2016;Supplemental Fig. S3A).…”

Rubisco Activases: AAA+ Chaperones Adapted to Enzyme Repair

“…Interestingly, the red alga Cyanidioschyzon merolae , containing Rubisco form ID (Figure 2B), has two cbbX genes, one nuclear-encoded and one plastid-encoded (Loganathan et al, 2016). It was recently shown that the functional CmRca is a 1:1 hetero-hexamer between nuclear- and plastid-encoded subunits (Loganathan et al, 2016).…”

“…It was recently shown that the functional CmRca is a 1:1 hetero-hexamer between nuclear- and plastid-encoded subunits (Loganathan et al, 2016). Both of these Rca subunits share 60–70% identity with RsRca.…”

“…Rca enzymes belong to the AAA+ protein superfamily (Neuwald et al, 1999) and use ATP-driven conformational changes to remodel Rubisco, thereby facilitating the release of the inhibitory sugar phosphates (Portis, 2003; Portis et al, 2008). Since the discovery, in the early 1980's, of the first Rca in a photosynthesis mutant of Arabidopsis thaliana (Portis and Salvucci, 2002), Rca enzymes have been identified in many photosynthetic organisms containing either green-type or red-type Rubiscos, from chemoautotrophic bacteria to higher plants (Mueller-Cajar et al, 2011; Sutter et al, 2015; Tsai et al, 2015; Loganathan et al, 2016) (Figure 2B). Although displaying considerable sequence variability, all Rca's share the core subunit architecture of AAA+ proteins, consisting of a N-terminal nucleotide binding domain with α/β Rossman fold and a C-terminal α-helical domain (Hanson and Whiteheart, 2005; Snider et al, 2008; Wendler et al, 2012).…”