A polymorphic motif in the small subunit of ADP-glucose pyrophosphorylase modulates interactions between the small and large subunits

SummaryThe heterotetrameric, allosterically regulated enzyme, adenosine-5¢-diphosphoglucose pyrophosphorylase(AGPase) catalyzes the rate-limiting step in starch synthesis. Despite vast differences in allosteric propertiesand a long evolutionary separation, heterotetramers of potato small subunit and maize large subunit haveactivity comparable to either parent in an Escherichia coli expression system. In contrast, co-expression ofmaize small subunit with the potato large subunit produces little activity as judged by in vivo activity stain. Topinpoint the region responsible for differential activity, we expressed chimeric maize/potato small subunits inE. coli. This identified a 55-amino acid motif of the potato small subunit that is critical for glycogen productionwhen expressed with the potato large subunit. Potato and maize small subunit sequences differ at five aminoacids in this motif. Replacement experiments revealed that at least four amino acids of maize origin wererequired to reduce staining. An AGPase composed of a chimeric potato small subunit containing the 55-aminoacid maize motif with the potato large subunit exhibited substantially less affinity for the substrates, glucose-1-phosphate and ATP and an increased Ka for the activator, 3-phosphoglyceric acid. Placement of the potatomotif into the maize small subunit restored glycogen synthesis with the potato large subunit. Hence, a smallpolymorphic motif within the small subunit influences both catalytic and allosteric properties by modulatingsubunit interactions.Keywords: starch synthesis, ADP-glucose pyrophosphorylase, maize endosperm, potato tuber, bacterialexpression, subunit interactions.IntroductionStarch has many uses in industry and is an important en-ergy source for both animals and humans. As adenosine-5¢-diphosphoglucose pyrophosphorylase (AGPase) catalyzes arate-limiting step in starch synthesis, this enzyme hasattracted wide interest for potential crop improvement.Results from several plants have proved the fundamentalimportance of AGPase in starch synthesis. Starch yieldsincrease in potato tubers (Stark et al., 1992), maize (Girouxet al., 1996), wheat (Smidansky et al., 2002) and rice seeds(Smidansky et al., 2003) of plants containing an AGPasewith decreased sensitivity to its physiological inhibitor.Plant AGPases are heterotetramers of two small and twolarge subunits. Although forms of each subunit sharesignificant sequence similarity, the small subunit is more