Phosphorylation of the cytosolic glucose-6-phosphate dehydrogenase isozyme AtG6PD6 in response to phosphate nutrition of the model plant Arabidopsis thaliana
Smith, Milena A.
Plants , Biochemistry , Phosphate , G6PD
Glucose-6-phosphate dehydrogenase (G6PD) is a tightly regulated enzyme that catalyzes the first committed step of the oxidative pentose phosphate pathway (OPPP). The OPPP plays a pivotal role in generating reducing power in the form of NADPH, and carbon skeletons (e.g., ribose-5-P) needed for anabolism and cell growth. Our recent phosphoproteomics study discovered that the cytosolic G6PD isozyme, AtG6PD6, became hyperphosphorylated at multiple N-terminal residues (i.e., Ser12, Thr13, and Ser18) 48 h following resupply of 2 mM phosphate (Pi) to Pi-starved (–Pi) Arabidopsis thaliana cell cultures. Although other phosphoproteomic studies have documented in vivo N-terminal phosphorylation of AtG6PD6 and its orthologs from other plant species, the functions and mechanisms of this phosphorylation event remain unknown. The aim of this thesis was to test the hypothesis that N-terminal phosphorylation activates AtG6PD6 to enhance OPPP flux during the rapid resumption of cell growth that follows Pi resupply to –Pi Arabidopsis. Immunoblotting with an anti-(pSer18 phosphosite-specific AtG6PD6) antibody confirmed that AtG6PD6 from Pi-resupplied, but not –Pi, Arabidopsis suspension cells and roots was in vivo phosphorylated at Ser18. This correlated with an approximate 54% increase in extractable G6PD activity following Pi-resupply to –Pi Arabidopsis seedlings (i.e., root extracts). Although the native enzyme is highly unstable in vitro, a partial purification of phosphorylated AtG6PD6 was achieved from Pi-replete suspension cells, with Vmax, Km(G6P), and S0.5(NADP+) values of 1.8 (µmol NADPH produced min-1) mg-1, 240 µM, and 38 µM, respectively. SDS-PAGE, immunoblotting, and analytical gel-filtration indicated the final preparation exists as a homotetramer composed of 59 kD subunits. Liquid chromatography-tandem mass spectroscopy analysis revealed the final preparation was a mixture of both cytosolic G6PD isozymes encoded by the Arabidopsis genome, namely AtG6PD5 and AtG6PD6. Following λ-phosphatase-mediated dephosphorylation of purified AtG6PD5/6, no differences in substrate saturation kinetics were detected, possibly owing to sub-stoichiometric phosphoryl group incorporation into the purified enzyme. Assessing the interplay between Pi nutrition and cytosolic G6PD phosphorylation may contribute to our understanding of post-translational OPPP control in plants, while identifying targets for developing Pi-efficient crop varieties. Such cultivars are urgently needed to alleviate agriculture’s over-reliance on massive applications of non-renewable and polluting Pi fertilizers.