Phosphorylation of chloroplast ribulose bisphosphate carboxylase/oxygenase small subunit by an envelope-bound protein kinase in situ

A new protein kinase of the cAMP independent type was found to be bound to the outer envelope membrane of spinach chloroplasts. While stimulated by Mg2+ and inhibited by ADP, the enzyme showed no response to conventional protein substrates and was essentially independent of pH in the physiological (pH 7 to 8) range. The new protein kinase phosphorylated the mature form of the small subunit of ribulose 1,5- bisphosphate carboxylase/oxygenase and, to a lesser extent, an unidentified 24-kDa polypeptide, both of which were bound to the outer envelope membrane. The results suggest that phosphorylation of cytoplasmically synthesized protein constituents of chloroplasts is involved in their transport through the chloroplast envelope membrane barrier

An enzyme synthesizing fructose 2,6-bisphosphate occurs in leaves and is regulated by metabolite effectors

AbstractAn enzyme catalyzing the ATP and fructose 6-phosphate-dependent synthesis of fructose 2,6-bisphosphate, a regulator of glycolysis and gluconeogenesis, has been identified and partially purified from plants, specifically the cytoplasmic fraction of spinach leaf parenchyma cells. The enzyme, designated fructose 6-phosphate, 2-kinase, showed no response to a protein phosphorylation system known to inhibit the corresponding enzyme in mammalian cells, but it responded strikingly to metabolite effectors (Pi, an activator/PGA, an inhibitor) through changes in substrate affinity and maximal velocity. The observed pattern of regulation suggests a role for chloroplasts in controlling cytoplasmic carbon processing

Metabolite-mediated catalyst conversion of PFK and PFP

Metabolites known to occur in the cytosol of photosynthetic leaf cells were found to mediate the reversible conversion of pyrophosphate—D-fructose-6-phosphate 1-phosphotransferase (PFP) to phosphofructokinase (PFK) in partially purified preparations from spinach leaves. Preincubation of PFP with fructose 2,6-bisphosphate, ATP or fructose 6-phosphate converted PFP to PFK. The reverse reaction (PFK → PFP) was promoted by UDP-glucose plus pyrophosphate. These conversions in catalytic capability were accompanied by changes in molecular mass and charge. The results are in accord with the view that the alterations in PFP and PFK activity, provisionally called ‘metabolite-mediated catalyst conversion’, represent a regulatory mechanism to direct left cytosolic carbon flux in either the biosynthetic or degradatory direction

Principal component analysis and biochemical characterization of protein and starch reveal primary targets for improving sorghum grain

Limited progress has been made on genetic improvement of the digestibility of sorghum grain because of variability among different varieties. In this study, we applied multiple techniques to assess digestibility of grain from 18 sorghum lines to identify major components responsible for variability. We also identified storage proteins and enzymes as potential targets for genetic modification to improve digestibility. Results from principal component analysis revealed that content of amylose and total starch, together with protein digestibility (PD), accounted for 94% of variation in digestibility. Control of amylose content is understood and manageable. Up-regulation of genes associated with starch accumulation is clearly a future target for improving digestibility. To identify proteins that might be targets for future modification, meal from selected lines was digested in vitro with pancreatin in parallel with pepsin and α-amylase. The %PD was influenced by both the nature of the protein matrix and protein body packaging. Owing to its ability to form oligomers, the 20 kDa γ-kafirin was more resistant to digestion than counterparts lacking this ability, making it a target for down-regulation. Greater understanding of interactions among the three traits identified by principal component analysis is needed for both waxy and non-waxy varieties

Unraveling thioredoxin-linked metabolic processes of cereal starchy endosperm using proteomics

AbstractApplication of a thiol-specific probe, monobromobimane, with proteomics and enzyme assays led to the identification of 23 thioredoxin targets in the starchy endosperm of mature wheat seeds (Triticum aestivum cv. Butte), almost all containing at least two conserved cysteines. The identified targets, 12 not known to be thioredoxin-linked, function in a spectrum of processes: metabolism (12 targets), protein storage (three), oxidative stress (three), protein degradation (two), protein assembly/folding (one) and unknown reactions (two). In addition to formulating metabolic pathways functional in the endosperm, the results suggest that thioredoxin acts in redox regulation throughout the life cycle of the seed

Unprecedented pathway of reducing equivalents in a diflavin-linked disulfide oxidoreductase

Flavoproteinsparticipateinawidevarietyofphysiologicallyrelevant processes that typically involve redox reactions. Within this protein superfamily, there exists a group that is able to transfer reducing equivalents from FAD to a redox-active disulfide bridge, which further reduces disulfide bridges in target proteins to regulate their structure and function. We have identified a previously undescribed type of flavin enzyme that is exclusive to oxygenic photosynthetic prokaryotes and that is based on the primary sequence that had been assigned as an NADPH-dependent thioredoxin reductase (NTR). However, our experimental data show that the protein does not transfer reducing equivalents from flavins to disulfides as in NTRs but functions in the opposite direction. High-resolution structures of the protein from Gloeobacter violaceus and Synechocystis sp. PCC6803 obtained by X-ray crystallography showed two juxtaposed FADmoleculespermonomerinredoxcommunicationwithanactive disulfide bridge in a variant of the fold adopted by NTRs. We have tentatively named the flavoprotein “DDOR” (diflavin-linked disulfide oxidoreductase) and propose that its activity is linked to a thiol-basedtransferofreducingequivalentsinbacterialmembranes. These findings expand the structural and mechanistic repertoire of flavoenzymes with oxidoreductase activity and pave the way to explore new protein engineering approaches aimed at designing redox-active proteins for diverse biotechnological applications.Spanish Ministerio de Economía, Industria y Competitividad BFU2016-80343-P, BIO2016-75634-