Dynamic photosynthetic labelling and carbon-positional mass spectrometry monitorin vivocarbon assimilation rates by ribulose-1,5-bisphosphate carboxylase

Abstract Ribulose-1,5-bisphosphate carboxylase/oxygenase (RUBISCO) is the most abundant enzyme and CO 2 bio-sequestration system on earth. Its in vivo activity is usually determined by 14 CO 2 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 13 CO 2 labelling allowed quantification of the 13 C fractional enrichment (E 13 C ) and molar carbon assimilation rates (A 13 C ) at carbon position 1-C of 3PGA by combination of E 13 C from carbon positions 2,3-C 2 and 1,2,3-C 3 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 Δgapdh1 and Δgapdh2 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 Δgapdh2 mutant and a CBB cycle modulating role of GAPDH1 under fluctuating CO 2 supply. RUBISCO activity in the CBB-deficient Δgapdh2 mutant can re-assimilate CO 2 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.