Exploring triosephosphate isomerase diversity in photosynthetic organisms

Abstract

<p><b>Triosephosphate isomerase (TPI) is one of the most well characterised enzymes in the BRaunschweig ENzyme DAtabase (BRENDA). TPI is widely investigated in the context of glycolysis/gluconeogenesis, however, in photosynthetic organisms TPI is also involved in the regeneration phase of the Calvin-Benson-Bassham (CBB) cycle. Cyanobacteria possess a single TPI involved in both pathways, however, plants and many algae possess two TPI isoforms, a cytoplasmic isoform and a chloroplastic isoform, that are involved in glycolysis/gluconeogenesis and the CBB cycle respectively. This TPI organisation provides an opportunity to investigate how photosynthesis and adaptation to the chloroplast has shaped the evolution of TPI. For instance, the TPI inhibitor, 2-phosphoglycolate (2PG) is produced in the chloroplast due to oxygenation activity of ribulose 1,5 bisphosphate carboxylase/oxygenase (rubisco).</b></p> <p>This project gains insight into TPI evolution by investigating the TPIs of three photosynthetic, and one derived non-photosynthetic, species. The organisms investigated were; the model cyanobacterium Synechocystis sp. PCC6803, the model plant Arabidopsis thaliana, the red alga Porphyra umbilicalis, and the non-photosynthetic parasitic plant Cuscuta australis. The TPIs from these organisms were characterised using structure/function and kinetic paradigms. </p> <p>The characterisation of Synechocystis and A. thaliana TPIs were used to establish workflow and to generate a baseline to compare the underexplored TPIs of C. australis and P. umbilicalis. </p> <p>The C. australis TPIs were investigated in the context of relaxed purifying selection resulting from adaptation of C. australis to parasitism and subsequent loss of photosynthesis. I reported that relaxed purifying selection has eroded the activity of C. australis TPIs compared to A. thalaina orthologues. Additionally, the structures of both C. australis cytoplasmic and chloroplast TPIs were solved by X-ray crystallography to identify where in the structure relaxed purifying selection has acted. </p> <p>The P. umbilicalis TPIs were investigated in the context of 2PG inhibition as the rubisco of red alga are infamous for their low rate of rubisco oxygenation activity. I have demonstrated that there is no difference in 2PG inhibition between P. umbilicalis and A. thaliana TPIs. However, unexpectedly I identified that the chloroplast TPI of P. umbilicalis is exquisitely sensitive to oxidation.</p>