Phosphoglucan-bound structure of starch phosphatase Starch Excess4 reveals the mechanism for C6 specificity

Abstract

Plants use the insoluble polyglucan starch as their primary glucose storage molecule. Reversible phosphorylation, at the C6 and C3 positions of glucose moieties, is the only known natural modification of starch and is the key regulatory mechanism controlling its diurnal breakdown in plant leaves. The glucan phosphatase Starch Excess4 (SEX4) is a position-specific starch phosphatase that is essential for reversible starch phosphorylation; its absence leads to a dramatic accumulation of starch in Arabidopsis, but the basis for its function is unknown. Here we describe the crystal structure of SEX4 bound to maltoheptaose and phosphate to a resolution of 1.65 Å. SEX4 binds maltoheptaose via a continuous binding pocket and active site that spans both the carbohydrate-binding module (CBM) and the dual-specificity phosphatase (DSP) domain. This extended interface is composed of aromatic and hydrophilic residues that form a specific glucan-interacting platform. SEX4 contains a uniquely adapted DSP active site that accommodates a glucan polymer and is responsible for positioning maltoheptaose in a C6-specific orientation. We identified two DSP domain residues that are responsible for SEX4 sitespecific activity and, using these insights, we engineered a SEX4 double mutant that completely reversed specificity from the C6 to the C3 position. Our data demonstrate that the two domains act in consort, with the CBM primarily responsible for engaging glucan chains, whereas the DSP integrates them in the catalytic site for position-specific dephosphorylation. These data provide important insights into the structural basis of glucan phosphatase site-specific activity and open new avenues for their biotechnological utilization.tarch is the primary carbohydrate storage molecule in plants and is an essential constituent of human and animal diets. Starch granules are composed of the glucose homopolymers amylose (10-25%) and amylopectin (75-90%) (1, 2). Amylose is a linear molecule formed from α-1,4-glycosidic-linked chains, whereas amylopectin is formed from α-1,4-glycosidic-linked chains with α-1,6-glycosidic-linked branches (3, 4). Adjacent amylopectin chains interact to form double helices that cause starch granules to be water insoluble, which is an essential feature for its function as a glucose storage molecule (1, 3, 5). However, the outer granular surface of transitory starch must be solubilized during nonphotosynthetic periods so that glycolytic enzymes can access and degrade starch glucans and meet the metabolic needs of the plant (6, 7). Plants regulate the solubility of the starch granular surface via reversible starch phosphorylation that results in a cyclic degradative process: phosphorylation by dikinases, degradation by starch hydrolyzing amylases, and dephosphorylation by phosphatases (1, 8-11). Phosphorylation of amylopectin chains causes helical unwinding and local solubilization of the outer starch granule (12-14). The local solubilization and helix unwinding permits degradation of surface, linear α-1,4 glucan chai...

Mechanistic Insights into Glucan Phosphatase Activity against Polyglucan Substrates

“…Cloning, Expression, and Purification of Recombinant Proteins-Wild type Arabidopsis thaliana SEX4 and LSF2 constructs were designed as previously described (28,57). Both constructs (⌬89-SEX4, ⌬78-LSF2) lack the chloroplast targeting peptide (cTP) along with residues up to the DSP recognition domain, and were subcloned into a pET-28b vector (Novagen) using NdeI and XhoI restriction sites to encode an N-terminal His 6 tag, a thrombin cleavage site, and SEX4 or LSF2.…”

“…LSF2 contains a cTP, DSP domain, and a CT motif (15). SEX4 preferentially dephosphorylates the C6-position of starch glucose and LSF2 exclusively dephosphorylates the C3-position (15,27,28). Arabidopsis sex4 mutants contain excess leaf starch, decreased plant growth, and an accumulation of shortchain phospho-oligosaccharides (19,29).…”

“…These differences in substrate specificity are driven, in large part, by the architecture of the DSP (53,54,56). Recent structural work has elucidated the primary mechanism underlying the method by which all three glucan phosphatases bind glucan chains and integrate them into the catalytic site (28,42,57). The glucan phosphatase DSP domains all contain aromatic/ hydrophobic residues that act as platforms to bind glucan chains at the active site (28,42,57).…”

“…In addition, each glucan phosphatase also employs an ancillary glucan-binding domain. The SEX4 structure revealed that its DSP and CBM domains form a continuous binding pocket and simultaneously engage the glucan chain (28). Conversely, the DSP and CBM in laforin are spatially separated and engage glucan chains independently (42).…”

Family 46 Carbohydrate-binding Modules Contribute to the Enzymatic Hydrolysis of Xyloglucan and β-1,3–1,4-Glucans through Distinct Mechanisms

“…Structural data revealed that CBM3c extends the catalytic site of the associated GH9 catalytic domains (22). More recent examples of CBMs modulating catalytic specificity is provided by a type C fructan-binding CBM66 that directs the cognate enzyme toward highly branched glucans rather than linear fructose polymers (24) and a CBM48 that contributes to substrate binding at the active site of a glucan phosphatase (25).…”