The ADP-glucose pyrophosphorylase from Melainabacteria: a comparative study between photosynthetic and non-photosynthetic bacterial sources

Until recently, all members of the cyanobacterial phylum were considered capable of performing oxygenic photosynthesis. This view has been questioned after the discovery of a group of presumed non-photosynthetic cyanobacteria named Melainabacteria. Using metagenomic data, we identified sequences encoding putative ADP-glucose pyrophosphorylase (EC 2.7.7.27, ADP-GlcPPase) from free-living and intestinal Melainabacteria. These genes were de novo synthesized and overexpressed in Escherichia coli. The purified recombinant proteins from the free-living and the intestinal Melainabacteria showed ADP-GlcPPase activity, with Vmax values of 2.3 and 7.1 U/mg, respectively. Both enzymes had similar affinities towards ATP (S0.5 ~0.3 mM) although the one from the intestinal source displayed a 6-fold higher affinity for glucose-1P. Both recombinant ADP-GlcPPases were sensitive to allosteric activation by glucose-6P (A0.5 ~0.3 mM), and to inhibition by Pi and ADP (I0.5 between 0.2 to 3 mM). Interestingly, the enzymes from Melainabacteria were insensitive to 3-phosphoglycerate, which is the principal activator of ADP-GlcPPases from photosynthetic cyanobacteria. To the best of our knowledge, this is the first biochemical characterization of an active enzyme from Melainabacteria, offering further data to discussions regarding their phylogenetic position. This work contributes to a better understanding regarding the evolution of allosteric mechanisms in ADP-GlcPPases, an essential enzyme for the synthesis of glycogen in prokaryotes and starch in plants.

Metabolite-level carbon-flow analysis reveals that starch synthesis from hexose is a limiting factor in a high-yielding rice cultivar

Understanding the limiting factors of grain filling is essential for the further improvement of rice (Oryza sativa L.) grain yields. The slower grain growth of Momiroman, a high-yielding rice cultivar, is not improved by increasing the carbon supply. Thus, a low sink activity, which is the metabolic activity of assimilate consumption/storage in sink organs, may be a limiting factor of grain filling. However, there is no metabolic evidence corroborating this hypothesis, partly because there is no consensus on how to define and quantify sink activity. In this study, we investigated the carbon flows, at a metabolite level, from photosynthesis in leaves to starch synthesis in grains of three high-yielding cultivars using a stable isotope of carbon, 13C. The large amount of newly fixed carbon assimilates in Momiroman was stored as hexose instead of being converted to starch. Additionally, the activity of ADP-glucose pyrophosphorylase and the expression of AGPS2b, encoding an ADP-glucose pyrophosphorylase protein, in the superior grains of Momiroman were lower than in the other two rice cultivars. Thus, the slower starch synthesis from hexose, which is partly explained by the low expression level of AGPS2b, may be the primarily metabolic reason for the lower sink activity in Momiroman. (199/200 words)