The Different Large Subunit Isoforms of Arabidopsis thaliana ADP-glucose Pyrophosphorylase Confer Distinct Kinetic and Regulatory Properties to the Heterotetrameric Enzyme

Abstract

ADP-glucose pyrophosphorylase catalyzes the first and limiting step in starch biosynthesis and is allosterically regulated by the levels of 3-phosphoglycerate and phosphate in plants. ADP-glucose pyrophosphorylases from plants are heterotetramers composed of two types of subunits (small and large). In this study, the six Arabidopsis thaliana genes coding for ADP-glucose pyrophosphorylase isoforms (two small and four large subunits) have been cloned and expressed in an Escherichia coli mutant deficient in ADP-glucose pyrophosphorylase activity. The co-expression of the small subunit APS1 with the different Arabidopsis large subunits (APL1, APL2, APL3, and APL4) resulted in heterotetramers with different regulatory and kinetic properties. Heterotetramers composed of APS1 and APL1 showed the highest sensitivity to the allosteric effectors as well as the highest apparent affinity for the substrates (glucose-1-phosphate and ATP), whereas heterotetramers formed by APS1 and APL2 showed the lower response to allosteric effectors and the lower affinity for the substrates. No activity was detected for the second gene coding for a small subunit isoform (APS2) annotated in the Arabidopsis genome. This lack of activity is possibly due to the absence of essential amino acids involved in catalysis and/or in the binding of glucose-1-phosphate and 3-phosphoglycerate. Kinetic and regulatory properties of the different heterotetramers, together with sequence analysis has allowed us to make a distinction between sink and source enzymes, because the combination of different large subunits would provide a high plasticity to ADP-glucose pyrophosphorylase activity and regulation. This is the first experimental data concerning the role that all the ADP-glucose pyrophosphorylase isoforms play in a single plant species. This phenomenon could have an important role in vivo, because different large subunits would confer distinct regulatory properties to ADP-glucose pyrophosphorylase according to the necessities for starch synthesis in a given tissue.

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

“…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). The unicellular algae branches of the tree for S and L subunits have not been characterized (Fig.…”

“…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 has been postulated to have a role in catalysis due to effects on apparent affinity of substrates (6), but this may be explained by allosteric effects.…”

“…In plants and green algae, the presence of two types of subunits expanded the sequence space to explore new roles. Recently, it was shown that L subunit isoforms from Arabidopsis had different properties based on the tissue expression (2,10). On the other hand, the presence of two types of subunits may have constrained each subunit to adopt complementary roles.…”

KONJAC1 and 2 Are Key Factors for GDP-Mannose Generation and Affect l-Ascorbic Acid and Glucomannan Biosynthesis in Arabidopsis

“…ADP-Glc pyrophosphorylases provide ADP-Glc for starch synthesis and can be divided into two clades, small (S) subunit and large (L) subunit clades. As reported for ADP-Glc pyrophosphorylase S1 of Arabidopsis, the S subunit plays a central role in the synthesis of ADP-Glc, whereas the L subunit has weak enzymatic activity and instead functions as regulatory protein to enhance the activity of the S subunits (Crevillén et al, 2003;Petreikov et al, 2010).…”

Isolation and characterization of cDNAs and genomic DNAs encoding ADP-glucose pyrophosphorylase large and small subunits from sweet potato

“…On the contrary, the expressions of LS genes are usually tissue-and development-specific. The expression patterns and regulatory properties of different LS isoforms have been comprehensively studied in tomato (Park and Chung 1998), Arabidopsis (Crevillén et al 2003(Crevillén et al , 2005, and rice , and the results of those studies strongly indicate that AGPase heterotetramers formed in different tissues or organs display distinct kinetic and regulatory properties to control starch biosynthesis. It is suggested that the LSs of agriculturally important crops such as potato and tomato, which have undergone significant selection under both domestication and natural selection, may have evolved novel modulatory characteristics (Petreikov et al 2010).…”