Two Arabidopsis ADP-Glucose Pyrophosphorylase Large Subunits (APL1 and APL2) Are Catalytic

Abstract

ADP-glucose (Glc) pyrophosphorylase (ADP-Glc PPase) catalyzes the first committed step in starch biosynthesis. Higher plant ADP-Glc PPase is a heterotetramer (a 2 b 2 ) consisting of two small and two large subunits. There is increasing evidence that suggests that catalytic and regulatory properties of the enzyme from higher plants result from the synergy of both types of subunits. In Arabidopsis (Arabidopsis thaliana), two genes encode small subunits (APS1 and APS2) and four large subunits (APL1-APL4). Here, we show that in Arabidopsis, APL1 and APL2, besides their regulatory role, have catalytic activity. Heterotetramers formed by combinations of a noncatalytic APS1 and the four large subunits showed that APL1 and APL2 exhibited ADP-Glc PPase activity with distinctive sensitivities to the allosteric activator (3-phosphoglycerate). Mutation of the Glc-1-P binding site of Arabidopsis and potato (Solanum tuberosum) isoforms confirmed these observations. To determine the relevance of these activities in planta, a T-DNA mutant of APS1 (aps1) was characterized. aps1 is starchless, lacks ADP-Glc PPase activity, APS1 mRNA, and APS1 protein, and is late flowering in long days. Transgenic lines of the aps1 mutant, expressing an inactivated form of APS1, recovered the wild-type phenotype, indicating that APL1 and APL2 have catalytic activity and may contribute to ADP-Glc synthesis in planta.ADP-Glc pyrophosphorylase (ADP-Glc PPase; EC 2.7.7.27) is a heterotetrameric enzyme that catalyzes the synthesis of ADP-Glc and inorganic pyrophosphate from Glc-1-P and ATP (Espada, 1962). ADP-Glc is the donor for glycogen synthesis in bacteria and starch synthesis in plants (Sivak and Preiss, 1998; Slattery et al., 2000). ADP-Glc PPase is an allosteric enzyme regulated by intermediates of the major pathway of carbon assimilation in the organism (Ballicora et al., , 2004. The activity of many higher plant ADP-Glc PPases is allosterically activated by 3-phosphoglycerate (3-PGA) and inhibited by inorganic phosphate (Preiss, 1973;Sivak and Preiss, 1998). Most bacterial enzymes are homotetramers composed of four identical subunits (a 4 ; Haugen et al., 1976;, while higher plant ADP-Glc PPases are heterotetramers composed of two closely related types of subunits (S 2 L 2 ; Morell et al., 1987;Okita et al., 1990;Preiss et al., 1991;Smith-White and Preiss, 1992;Ballicora et al., 2004). In higher plants, a number of studies suggest that the regulatory properties of ADP-Glc PPase are a product of synergistic interactions between the two types of subunits (Ballicora et al., 1998; Slattery et al., 2000;Cross et al., 2004;Hwang et al., 2005;Ventriglia et al., 2007). Besides, recent evidence suggests that some L subunits may have catalytic activity or influence the net catalysis of the enzyme (Burger et al., 2003). It has been reported that potato (Solanum tuberosum) tuber L subunit can be turned catalytically active by mutagenesis (Ballicora et al., 2005;Hwang et al., 2008) and that mutation of potato tuber L subunit (PLS) amino acid P44 (number...

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.…”

“…To observe potential catalysis coming from OtaL without interference, we used a catalytically defective OtaS for the co-expression. A similar strategy was previously used to study the roles of subunits in Arabidopsis and potato tuber ADP-Glc PPases (4,5). In OtaS, we chose to replace Asp-148 with alanine to obtain a catalytically defective S subunit.…”

“…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.…”

The Sink-Specific Plastidic Phosphate Transporter PHT4;2 Influences Starch Accumulation and Leaf Size in Arabidopsis

“…Starch-deficient mutants do not have abnormally large leaves (Ventriglia et al, 2008), suggesting that reduced starch accumulation is unlikely to be causal for the increase in cell proliferation underlying the pht4;2 leafsize phenotype. However, starch accumulation in such mutants is affected throughout the entire plant, so comparisons may be misleading if the leaf-size phenotype is related to carbon balance between source and sink organs.…”

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

“…Numerous studies using various genetic, mutagenic, and biochemical strategies have strongly suggested that the SS has both catalytic and regulatory activities, and that the LS is mainly responsible for modulating the allosteric regulatory properties of the SS in the heterotetrameric enzyme (Ballicora et al 1995;Salamone et al 2000;Frueauf et al 2003;Hwang et al 2006). Although the LS is usually non-catalytic, recent studies have demonstrated that recombinant LSs (mutated or natural) show defective catalytic activity, both alone and in the presence of the SS in vitro Ventriglia et al 2008;Hwang et al 2008;Petreikov et al 2010). However, the binding of LS and SS to form a heterotetramer is essential for the enzyme to function efficiently in plants.…”