Plant AGPase activity can be modulated by several mechanisms: allosteric regulation by small effecter molecules, thermal inactivation, and reductive activation. Both subunit types evolved from a common progenitor ( Georgelis et al., 2007) and each exhibits a defective catalytic and/or regulatory activity when expressed alone in Escherichia coli ( Iglesias et al., 1993 Burger et al., 2003 Hwang et al., 2008). Plant AGPases possess a heterotetrameric structure composed of two small subunits (SS) and two large subunits (LS). Moreover, null AGPase mutants showed decreased starch content ( Johnson et al., 2003) while desensitization of AGPase activity to Pi inhibition led to increased seed yield ( Giroux et al., 1996 Smidansky et al., 2003 Cross et al., 2004). Transgenic cereals over-expressing modified forms of AGPase showed not only an enhanced rate of starch synthesis but increased final grain yield ( Stark et al., 1992 Giroux et al., 1996 Sakulsingharoj et al., 2004 Obana et al., 2006 Gibson et al., 2011). Evidence from previous studies indicate that AGPase catalyzes a major limiting step in starch synthesis. AGPase catalyzes the initial step in the starch biosynthetic pathway by converting ATP and О±-glucose-1-posphate (Glc1P) to ADP-glucose and pyrophosphate (PPi).

ADP-Glucose pyrophosphorylase (AGPase) plays an essential role in glycogen biosynthesis in bacteria ( Preiss, 1984, 1996) and is ubiquitous in starch-synthesizing plant tissues ( Lee et al., 2007). Starch is a major component in many plant seed endosperms, accounting for 56вЂ“74% of the available carbohydrates in the grain of most food crops including cereals ( Koehler and Wieser, 2013). Our molecular dynamic simulation analysis suggests that introduction of the QTC motif and the е…ѓ79F mutation improves enzyme heat stability by stabilizing their backbone structures possibly due to the increased number of H-bonds between the small subunits and increased intermolecular interactions between the two SSs and two LSs at elevated temperature. Modeled structure of this mutant enzyme revealed that this highly conserved leucine residue is located in the enzymeвЂ™s regulatory pocket that provides interaction sites for activators and inhibitors. All six lines contained a е…ѓ79F substitution, which conferred enhanced glycogen production in bacteria and increased heat stability. Random mutagenesis of S2b QTC produced six mutant lines with elevated production of glycogen in bacteria. While the QTC AGPase exhibited approximately fourfold increase in 3-PGA sensitivity, its substrate affinities were largely unchanged. The QTC enzyme showed moderate heat stability but was not as stable as the potato AGPase. Introduction of a QTC motif into S2b created an N-terminal disulfide linkage that was cleaved by dithiothreitol reduction. The S2b assembled with the mosaic L2-pLS subunit showed 3-fold higher sensitivity to 3-PGA than L2/S2b, whereas the counterpart mosaic pLS-L2/S2b showed 225-fold lower sensitivity. However, assembly of the rice L2 with the potato tuber SS (pSS) showed significantly increased heat stability comparable to the heat-stable potato pLS/pSS. Bacterial expression of the rice S2b with the rice L2, potato tuber LS (pLS), or with the mosaic rice-potato large subunits, L2-pLS and pLS-L2, produced heat-sensitive recombinant enzymes, which retained less than 10% of their enzyme activities after 5 min incubation at 55вЊ. To increase the heat tolerance of the rice enzyme, we engineered two dominant AGPase subunits expressed in developing endosperm, the large (L2) and small (S2b) subunits of the cytosol-specific AGPase. ADP-glucose pyrophosphorylase (AGPase) is among the starch biosynthetic enzymes severely affected by heat stress during seed maturation. Rice grains accumulate starch as their major storage reserve whose biosynthesis is sensitive to heat. 3Distributed Information Centre (DIC), Department of Agricultural Biotechnology, Assam Agricultural University, Jorhat, India.2Department of Agricultural Biotechnology, Assam Agricultural University, Jorhat, India.1Institute of Biological Chemistry, Washington State University, Pullman, WA, United States.Seon-Kap Hwang 1вЂ , Salvinder Singh 2вЂ , Jitendra Maharana 3вЂ , Samhita Kalita 2, Aytug Tuncel 1, Tanmayee Rath 3, Debashish Panda 3, Mahendra Kumar Modi 2 and Thomas W.

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