Dynamic photosynthetic labelling and carbon-positional mass spectrometry monitor in vivo carbon assimilation rates by ribulose-1,5-bisphosphate carboxylase.

AbstractRibulose-1,5-bisphosphate carboxylase/oxygenase (RUBISCO) is the most abundant enzyme and CO2 bio-sequestration system on earth. Its in vivo activity is usually determined by 14CO2 incorporation into 3-phosphoglycerate (3PGA). The radiometric analysis of 3PGA does not distinguish carbon positions. Hence, RUBISCO activity that fixes carbon into 1-C position of 3PGA and Calvin-Benson-Bassham (CBB) cycle activities that redistribute carbon into its 2-C and 3-C positions are not resolved. This study aims to provide technology that differentiates between these activities. In source fragmentation of gas chromatography-mass spectrometry (GC- MS) enables paired isotopologue distribution analyses of fragmented substructures and the complete metabolite structure. GC-MS measurements after dynamic photosynthetic 13CO2 labelling allowed quantification of the 13C fractional enrichment (E13C) and molar carbon assimilation rates (A13C) at carbon position 1-C of 3PGA by combination of E13C from carbon positions 2,3-C2 and 1,2,3-C3 with quantification of 3PGA concentrations. We validated the procedure using two GC-time of flight (TOF)-MS instruments, operated at nominal or high mass resolution and tested expected positional labelling of 3PGA by in vivo glycolysis of positional labelled glucose isotopomers. Application to{Delta} gapdh1 and{Delta} gapdh2 mutants of the highly divergent glyceraldehyde-3-phosphate dehydrogenases (GAPDH) from Synechocystis sp. PCC 6803 revealed full inactivation of the CBB cycle with maintained RUBISCO activity in the{Delta} gapdh2 mutant and a CBB cycle modulating role of GAPDH1 under fluctuating CO2 supply. RUBISCO activity in the CBB-deficient{Delta} gapdh2 mutant can re-assimilate CO2 released by catabolic pathways. We suggest that RUBISCO activity in Synechocystis can scavenge carbon loss through the pentose phosphate pathway or other cellular decarboxylation reactions.