Mutagenesis of the Glucose-1-Phosphate-Binding Site of Potato Tuber ADP-Glucose Pyrophosphorylase1

Fu, Yingbin; Ballícora, Miguel A.; Preiss, Jack

doi:10.1104/pp.117.3.989

Cited by 50 publications

(47 citation statements)

References 35 publications

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“…We found that the model contains all residues in the active site that were previously found to be critical. Residues with a Glc-1-P binding role in the E. coli enzyme (Glu-194, Lys-195, Ser-212, Tyr-216, Asp-239, Phe-240, Trp-274, and Asp-276) (33,34) and in the StuS subunit (Lys-198 and Asp-252, also studied in E. coli) (32,35) were present in the corresponding positions (Fig. 2).…”

Section: Resultsmentioning

confidence: 90%

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Ostreococcus tauri ADP-glucose Pyrophosphorylase Reveals Alternative Paths for the Evolution of Subunit Roles

Kuhn¹,

Falaschetti²,

Ballícora

2009

Journal of Biological Chemistry

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ADP-glucose pyrophosphorylase controls starch synthesis in plants and is an interesting case to study the evolution and differentiation of roles in heteromeric enzymes. It includes two homologous subunits, small (S) and large (L), that originated from a common photosynthetic eukaryotic ancestor. In present day organisms, these subunits became complementary after loss of certain roles in a process described as subfunctionalization. For instance, the potato tuber enzyme has a noncatalytic L subunit that complements an S subunit with suboptimal allosteric properties. To understand the evolution of catalysis and regulation in this family, we artificially synthesized both subunit genes from the unicellular alga Ostreococcus tauri. This is among the most ancient species in the green lineage that diverged from the ancestor of all green plants and algae. After heterologous gene expression, we purified and characterized the proteins. The O. tauri enzyme was not redox-regulated, suggesting that redox regulation of ADP-glucose pyrophosphorylases appeared later in evolution. The S subunit had a typical low apparent affinity for the activator 3-phosphoglycerate, but it was atypically defective in the catalytic efficiency (V max /K m ) for the substrate Glc-1-P. The L subunit needed the S subunit for soluble expression. In the presence of a mutated S subunit (to avoid interference), the L subunit had a high apparent affinity for 3-phosphoglycerate and substrates suggesting a leading role in catalysis. Therefore, the subfunctionalization of the O. tauri enzyme was different from previously described cases. To the best of our knowledge, this is the first biochemical description of a system with alternative subfunctionalization paths.Starch synthesis in photosynthetic eukaryotes such as higher plants and unicellular algae is controlled by a heterotetrameric ADP-glucose pyrophosphorylase (ADP-Glc PPase, 4 EC 2.7.7.27). This enzyme catalyzes the reaction of ATP and Glc-1-P to form ADP-glucose (ADP-Glc) and PP i , and it is primarily activated by 3-phosphoglycerate (3-PGA) and inhibited by P i . In photosynthetic eukaryotes the enzyme includes two distinct S and L homologous subunits (S 2 L 2 or ␣ 2 ␤ 2 ), but in photosynthetic bacteria the enzyme is a homotetramer (␣ 4 ). There has been debate regarding the role of the different subunits of ADP-Glc PPase. The S subunit homotetramer from potato (Solanum tuberosum) tuber (StuS), Arabidopsis thaliana (APS1), and barley (Hordeum vulgare) endosperm have a catalytic function with defective regulatory properties (1-3). It is not clear if there is a set of universal roles for the L subunit in plants. The L subunit from potato tuber (StuL) is catalytically deficient and plays more of a regulatory role by modifying the apparent affinity of the S subunit toward allosteric regulators (1, 4). On the other hand, the Arabidopsis APL1 and APL2 isoforms have both catalytic and regulatory functions, whereas the APL3 and APL4 isoforms behave like StuL (2, 5). The maize (Zea mays) endosperm L subunit...

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Section: Resultsmentioning

confidence: 90%

“…1), including group I to IV and unicellular algae (4). Previously, L subunits from group III (StuL, APL3, and APL4) were shown to have a regulatory function with a defective catalytic role (2,4,5,31,32). On the other hand, isoforms APL1 and APL2 (groups I and II) were catalytic with a critical role in regulation (2,5).…”

Section: Resultsmentioning

confidence: 99%

Ostreococcus tauri ADP-glucose Pyrophosphorylase Reveals Alternative Paths for the Evolution of Subunit Roles

Kuhn¹,

Falaschetti²,

Ballícora

2009

Journal of Biological Chemistry

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“…The small subunits are mainly responsible for the catalytic properties whereas the large subunits are of regulatory importance (42). The two isoforms of the small AGPase subunit, for which peptides identical to published rice sequences were identified (refs.…”

Section: Resultsmentioning

confidence: 99%

Proteomic survey of metabolic pathways in rice

Koller

Washburn

Lange

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

373

224

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A systematic proteomic analysis of rice (Oryza sativa) leaf, root, and seed tissue using two independent technologies, two-dimensional gel electrophoresis followed by tandem mass spectrometry and multidimensional protein identification technology, allowed the detection and identification of 2,528 unique proteins, which represents the most comprehensive proteome exploration to date. A comparative display of the expression patterns indicated that enzymes involved in central metabolic pathways are present in all tissues, whereas metabolic specialization is reflected in the occurrence of a tissue-specific enzyme complement. For example, tissuespecific and subcellular compartment-specific isoforms of ADPglucose pyrophosphorylase were detected, thus providing proteomic confirmation of the presence of distinct regulatory mechanisms involved in the biosynthesis and breakdown of separate starch pools in different tissues. In addition, several previously characterized allergenic proteins were identified in the seed sample, indicating the potential of proteomic approaches to survey food samples with regard to the occurrence of allergens.

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“…The small subunit seemed to have all of the catalytic activity of the heterotetramer, because the large subunit expressed alone did not show ADP-Glc PPase activity (Iglesias et al, 1993;Ballicora et al, 1995). Sitedirected mutagenesis experiments on the Glc-1-P site confirmed the idea that the role of the large subunit on catalysis is minimal (Fu et al, 1998). We propose that the main role of the large subunit is to interact with the small subunit and modulate the properties of the activator site.…”

Section: Discussionmentioning

confidence: 99%

ADP-Glucose Pyrophosphorylase from Potato Tubers. Site-Directed Mutagenesis Studies of the Regulatory Sites1

Ballícora

Nesbitt

et al. 1998

Plant Physiology

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The apparent affinity for the inhibitor, phosphate, decreased greater than 400-fold. Mutation of Lys-441 to glutamic acid showed even larger effects. When Lys-417 and Lys-455 on the large subunit were mutated to alanine, the phosphate inhibition was not altered and the apparent affinity for the activator decreased only 9-and 3-fold, respectively. Mutations of these residues to glutamic acid only decreased the affinity for the activator 12-and 5-fold, respectively. No significant changes were observed on other kinetic constants for the substrates ADP-Glc, pyrophosphate, and Mg 2؉ . These data indicate that Lys-404 and Lys-441 on the small subunit are more important for the regulation of the ADP-Glc pyrophosphorylase than their homologous residues in the large subunit.

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Mutagenesis of the Glucose-1-Phosphate-Binding Site of Potato Tuber ADP-Glucose Pyrophosphorylase1

Cited by 50 publications

References 35 publications

Ostreococcus tauri ADP-glucose Pyrophosphorylase Reveals Alternative Paths for the Evolution of Subunit Roles

Ostreococcus tauri ADP-glucose Pyrophosphorylase Reveals Alternative Paths for the Evolution of Subunit Roles

Proteomic survey of metabolic pathways in rice

ADP-Glucose Pyrophosphorylase from Potato Tubers. Site-Directed Mutagenesis Studies of the Regulatory Sites1

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