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Resurrecting the Regulatory Properties of the Ostreococcus tauri ADP-Glucose Pyrophosphorylase Large Subunit.

ABSTRACT: ADP-glucose pyrophosphorylase (ADP-Glc PPase) catalyzes the first committed step for the synthesis of glycogen in cyanobacteria and starch in green algae and plants. The enzyme from cyanobacteria is homotetrameric (?4), while that from green algae and plants is heterotetrameric (?2?2). These ADP-Glc PPases are allosterically regulated by 3-phosphoglycerate (3PGA, activator) and inorganic orthophosphate (Pi, inhibitor). Previous studies on the cyanobacterial and plant enzymes showed that 3PGA binds to two highly conserved Lys residues located in the C-terminal domain. We observed that both Lys residues are present in the small (?) subunit of the Ostreococcus tauri enzyme; however, one of these Lys residues is replaced by Arg in the large (?) subunit. In this work, we obtained the K443R and R466K mutants of the O. tauri small and large subunits, respectively, and co-expressed them together or with their corresponding wild type counterparts. Our results show that restoring the Lys residue in the large subunit enhanced 3PGA affinity, whereas introduction of an Arg residue in the small subunit reduced 3PGA affinity of the heterotetramers. Inhibition kinetics also showed that heterotetramers containing the K443R small subunit mutant were less sensitive to Pi inhibition, but only minor changes were observed for those containing the R466K large subunit mutant, suggesting a leading role of the small subunit for Pi inhibition of the heterotetramer. We conclude that, during evolution, the ADP-Glc PPase large subunit from green algae and plants acquired mutations in its regulatory site. The rationale for this could have been to accommodate sensitivity to particular metabolic needs of the cell or tissue.

SUBMITTER: Figueroa CM

PROVIDER: S-EPMC6218581 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Resurrecting the Regulatory Properties of the Ostreococcus tauri ADP-Glucose Pyrophosphorylase Large Subunit.

Figueroa Carlos M CM Kuhn Misty L ML Hill Benjamin L BL Iglesias Alberto A AA Ballicora Miguel A MA

Frontiers in plant science 20181030

ADP-glucose pyrophosphorylase (ADP-Glc PPase) catalyzes the first committed step for the synthesis of glycogen in cyanobacteria and starch in green algae and plants. The enzyme from cyanobacteria is homotetrameric (α4), while that from green algae and plants is heterotetrameric (α2β2). These ADP-Glc PPases are allosterically regulated by 3-phosphoglycerate (3PGA, activator) and inorganic orthophosphate (Pi, inhibitor). Previous studies on the cyanobacterial and p ...[more]

PMID: 30425723

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Project description:ADP-glucose pyrophosphorylase from Firmicutes is encoded by two genes (glgC and glgD) leading to a heterotetrameric protein structure, unlike those in other bacterial phyla. The enzymes from two groups of Firmicutes, Bacillales and Lactobacillales, present dissimilar kinetic and regulatory properties. Nevertheless, no ADP-glucose pyrophosphorylase from Clostridiales, the third group in Firmicutes, has been characterized. For this reason, we cloned the glgC and glgD genes from Ruminococcus albus Different quaternary forms of the enzyme (GlgC, GlgD, and GlgC/GlgD) were purified to homogeneity and their kinetic parameters were analyzed. We observed that GlgD is an inactive monomer when expressed alone but increased the catalytic efficiency of the heterotetramer (GlgC/GlgD) compared to the homotetramer (GlgC). The heterotetramer is regulated by fructose-1,6-bisphosphate, phosphoenolpyruvate, and NAD(P)H. The first characterization of the Bacillales enzyme suggested that heterotetrameric ADP-glucose pyrophosphorylases from Firmicutes were unregulated. Our results, together with data from Lactobacillales, indicate that heterotetrameric Firmicutes enzymes are mostly regulated. Thus, the ADP-glucose pyrophosphorylase from Bacillales seems to have distinctive insensitivity to regulation.IMPORTANCE The enzymes involved in glycogen synthesis from Firmicutes have been less characterized in comparison with other bacterial groups. We performed kinetic and regulatory characterization of the ADP-glucose pyrophosphorylase from Ruminococcus albus Our results showed that this protein that belongs to different groups from Firmicutes (Bacillales, Lactobacillales, and Clostridiales) presents dissimilar features. This study contributes to the understanding of how this critical enzyme for glycogen biosynthesis is regulated in the Firmicutes group, whereby we propose that these heterotetrameric enzymes, with the exception of Bacillales, are allosterically regulated. Our results provide a better understanding of the evolutionary relationship of this enzyme family in Firmicutes.

Project description:BackgroundLandoltia punctata can be used as renewable and sustainable biofuel feedstock because it can quickly accumulate high starch levels. ADP-glucose pyrophosphorylase (AGPase) catalyzes the first committed step during starch biosynthesis in higher plants. The heterotetrameric structure of plant AGPases comprises pairs of large subunits (LSs) and small subunits (SSs). Although several studies have reported on the high starch accumulation capacity of duckweed, no study has explored the underlying molecular accumulation mechanisms and their linkage with AGPase. Therefore, this study focused on characterizing the roles of different L. punctate AGPases. Methodology. Expression patterns of LpAGPs were determined through comparative transcriptome analyses, followed by coexpressing their coding sequences in Escherichia coli, Saccharomyces cerevisiae, Arabidopsis thaliana, and Nicotiana tabacum.ResultsComparative transcriptome analyses showed that there are five AGPase subunits encoding cDNAs in L. punctata (LpAGPS1, LpAGPS2, LpAGPL1, LpAGPL2, and LpAGPL3). Nutrient starvation (distilled water treatment) significantly upregulated the expression of LpAGPS1, LpAGPL2, and LpAGPL3. Coexpression of LpAGPSs and LpAGPLs in Escherichia coli generated six heterotetramers, but only four (LpAGPS1/LpAGPL3, LpAGPS2/LpAGPL1, LpAGPS2/LpAGPL2, and LpAGPS2/LpAGPL3) exhibited AGPase activities and displayed a brownish coloration upon exposure to iodine staining. Yeast two-hybrid and bimolecular fluorescence complementation (BiFC) assays validated the interactions between LpAGPS1/LpAGPL2, LpAGPS1/LpAGPL3, LpAGPS2/LpAGPL1, LpAGPS2/LpAGPL2, and LpAGPS2/LpAGPL3. All the five LpAGPs were fusion-expressed with hGFP in Arabidopsis protoplasts, and their green fluorescence signals were uniformly localized in the chloroplast, indicating that they are plastid proteins.ConclusionsThis study uncovered the cDNA sequences, structures, subunit interactions, expression patterns, and subcellular localization of AGPase. Collectively, these findings provide new insights into the molecular mechanism of fast starch accumulation in L. punctata.

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Resurrecting the Regulatory Properties of the Ostreococcus tauri ADP-Glucose Pyrophosphorylase Large Subunit.

Publications

Resurrecting the Regulatory Properties of the <i>Ostreococcus tauri</i> ADP-Glucose Pyrophosphorylase Large Subunit.

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