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Starch synthases (SSs) are responsible for depositing the majority of glucoses in starch. Structural knowledge on these enzymes that is available from the crystal structures of rice granule bound starch synthase (GBSS) and barley SSI provides incomplete information on substrate binding and active site architecture. Here we report the crystal structures of the catalytic domains of SSIV from Arabidopsis thaliana, of GBSS from the cyanobacterium CLg1 and GBSSI from the glaucophyte Cyanophora paradoxa, with all three bound to ADP and the inhibitor acarbose. The SSIV structure illustrates in detail the modes of binding for both donor and acceptor in a plant SS. CLg1GBSS contains, in the same crystal structure, examples of molecules with and without bound acceptor, which illustrates the conformational changes induced upon acceptor binding that presumably precede catalytic activity. With structures available from several isoforms of plant and non-plant SSs, as well as the closely related bacterial glycogen synthases, we analyze, at the structural level, the common elements that define a SS, the elements that are necessary for substrate binding and singularities of the GBSS family that could underlie its processivity. While the phylogeny of the SSIII/IV/V has been recently discussed, we now further report the detailed evolutionary history of the GBSS/SSI/SSII type of SSs enlightening the origin of the GBSS enzymes used in our structural analysis.

The production of starch is essential for human nutrition and represents a major metabolic flux in the biosphere. The biosynthesis of starch in storage organs like barley endosperm operates via two main pathways using different substrates: starch synthases use ADP-glucose to produce amylose and amylopectin, the two major components of starch, whereas starch phosphorylase (Pho1) uses glucose-1-phosphate (G1P), a precursor for ADP-glucose production, to produce α-1,4 glucans. The significance of the Pho1 pathway in starch biosynthesis has remained unclear. To elucidate the importance of barley Pho1 (HvPho1) for starch biosynthesis in barley endosperm, we analyzed HvPho1 protein production and enzyme activity levels throughout barley endosperm development and characterized structure-function relationships of HvPho1. The molecular mechanisms underlying the initiation of starch granule biosynthesis, that is, the enzymes and substrates involved in the initial transition from simple sugars to polysaccharides, remain unclear. We found that HvPho1 is present as an active protein at the onset of barley endosperm development. Notably, purified recombinant protein can catalyze the de novo production of α-1,4-glucans using HvPho1 from G1P as the sole substrate. The structural properties of HvPho1 provide insights into the low affinity of HvPho1 for large polysaccharides like starch or amylopectin. Our results suggest that HvPho1 may play a role during the initiation of starch biosynthesis in barley.