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

Significance

Starch is the main carbohydrate storage molecule in plants and is ubiquitous in human life. Reversible starch phosphorylation is the key regulatory event in starch catabolism. Starch Excess4 (SEX4) preferentially dephosphorylates the C6 position of starch glucose and its absence results in a dramatic accumulation of leaf starch. We present the structure of SEX4 bound to a phosphoglucan product, define its mechanism of specific activity, and reverse its specificity to the C3 position via mutagenesis. The ability to control starch phosphorylation has direct applications in agriculture and industrial uses of starch. These insights into SEX4 structure and function provide a foundation to control reversible phosphorylation and produce designer starches with tailored physiochemical properties and potentially widespread impacts.

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 site-specific 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.

Starch 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 chains by β-amylase, which sequentially removes maltosyl units from the nonreducing end (1, 8, 15). Although glucan phosphorylation of the starch surface is necessary for degradation, the removal of these phosphate groups is required because β-amylase is unable to degrade past the phosphate (6, 15–17). Therefore, glucan phosphatases must release phosphate from starch to reset the degradation cycle, allowing processive starch degradation (8, 16).

Recent studies have established that plants use a two-enzyme system for both starch phosphorylation and dephosphorylation. α-Glucan water dikinase phosphorylates the hydroxyl group of starch glucose at the C6 position. This event triggers phosphorylation of the hydroxyl group at the C3 position by phosphoglucan water dikinase (18–21). Similarly, two glucan phosphatases release phosphate from starch. Starch Excess4 (SEX4) preferentially dephosphorylates the C6 position of starch glucose and Like Sex Four2 (LSF2) exclusively dephosphorylates the C3 position (22–26). SEX4 activity is essential for starch catabolism and its mutation in Arabidopsis leads to an excess of leaf starch, a decrease in plant growth, and an accumulation of soluble phosphoglucans produced by the activity of α-amylase 3 and the debranching enzyme isoamylase 3 (16, 25, 27). Conversely, lsf2 mutant Arabidopsis plants display normal levels of leaf starch and plant growth, but the starch contains increased levels of C3-phosphate (22). The difference in plant vitality between sex4 and lsf2 mutants is likely due to SEX4 possessing some compensatory C3-position phosphatase activity (22). Cumulatively, the process of reversible phosphorylation requires the concerted activity of dikinases and phosphatases with SEX4 activity being essential for normal patterns of starch metabolism and plant growth.

Glucan phosphatases are members of the protein tyrosine phosphatase (PTP) superfamily characterized by a conserved Cx₅R catalytic motif (24, 28, 29). The glucan phosphatases belong to a heterogeneous subset of PTPs called dual-specificity phosphatases (DSPs), with some DSPs dephosphorylating p-Tyr and p-Ser/Thr residues of proteinaceous substrates and other DSPs dephosphorylating lipids, nucleic acids, or glucans (30–32). In addition to SEX4 and LSF2, a glucan phosphatase called laforin has been identified that dephosphorylates glycogen and influences its solubility in vertebrates (24, 33, 34). Loss of laforin function in humans results in a fatal, neurodegenerative epilepsy called Lafora disease (35–37). Due to their critical function in complex carbohydrate metabolism, understanding the structural basis of glucan phosphatase activity is of particular interest. Toward this goal, we previously determined the ligand-free structure of SEX4 and identified an extensive interdomain interface between its DSP domain and carbohydrate-binding module (CBM) that is maintained in part by a previously unrecognized C-terminal (CT) motif (38). However, the structural mechanism for domain coupling, glucan interaction, and specific C6 dephosphorylation in SEX4 activity is unclear.

Starch granule solubilization depends on phosphorylation of starch glucose on the hydroxyl group at both the C6 and C3 positions (6, 13). These phosphorylation events are critical for normal transitory starch degradation, but also directly impact the melting temperature, viscosity, and hydration of starch in industrial settings (39, 40). Developing a means to manipulate starch phosphorylation patterns via enzymatic modification is relevant to agricultural and industrial applications that use starch as a feedstock (9, 12, 14, 41). Therefore, understanding the basis for the site specificity of glucan phosphatases is of particular interest. We recently determined the structure of LSF2 with a glucan bound in a C3-specific orientation and identified unique noncatalytic surface-binding sites (SBSs) not found in other glucan phosphatases (26). SEX4 lacks SBSs and preferentially dephosphorylates the C6 position. The present study was designed to define the fundamental basis for SEX4 substrate binding and understand preferential C6-position specificity in SEX4.

Herein, we elucidate the structural mechanism of SEX4-specific activity by determining the structure of SEX4 bound to the phosphoglucan products maltoheptaose and phosphate. SEX4 engages glucan chains via an extended interface of aromatic and hydrophilic residues that spans the CBM and DSP domains. Moreover, the SEX4 CBM is primarily responsible for glucan binding whereas the SEX4 DSP active site is uniquely adapted to engage the phosphoglucan substrate, positioning it in a C6-specific orientation. Structure-guided mutagenesis of DSP active-site residues resulted in a complete reversal from C6 to preferential C3 dephosphorylation by SEX4. Cumulatively, this study establishes the molecular basis for both SEX4 substrate engagement and SEX4 specificity and provides a method for engineering glucan phosphatase activity with modified site specificity.

Results

Structure of SEX4 Bound to Maltoheptaose and Phosphate.

The structure of the glucan phosphatase Arabidopsis thaliana SEX4 [residues 90–379, C198S (inactive mutant)], with maltoheptaose and phosphate bound in the active site, was determined to a resolution of 1.65 Å (Fig. 1A and Table 1). The crystallized SEX4 construct contains the catalytic DSP domain, the CBM, and CT motif. Maltoheptaose is composed of seven glucose moieties with α-1,4-glycosidic linkages, and clear electron density allowed the modeling of six glucose units of the maltoheptaose chain (Fig. 1B). The maltoheptaose chain is located within an extended pocket that spans the DSP and CBM domains, with Glc1 located at the DSP and Glc6 located at the CBM (numbered from nonreducing to reducing end). In addition, a single phosphate molecule was found within the catalytic site (PTP loop), directly below Glc2, at a distance of 2.5 Å from the catalytic residue S(C)198. The DSP–CBM pocket is ∼9 Å deep and ∼33 Å long with a total contact area of 610 Ų. Of this contact area, 40% of the interactions occur via the CBM domain and 60% via the DSP. Together, maltoheptaose and phosphate represent the product of dephosphorylation of the endogenous SEX4 phosphoglucan substrate, thus demonstrating the structural basis for SEX4–phosphoglucan interactions.

Fig. 1.

Crystal structure of At-SEX4 bound to a glucan chain and phosphate. (A) Surface/ribbon diagram of At-SEX4 [residues 90–379, C198S (catalytically inactive mutant)] bound to maltoheptaose (green) and phosphate (orange) determined to a resolution of 1.65 Å. The SEX4 structure contains the DSP domain (blue) with the catalytic site (red), the CBM (pink), and the CT motif (tan). The maltoheptaose chain (green) is located in an extended pocket spanning the CBM and DSP domains, and a single phosphate molecule is located at the base of the catalytic site directly beneath Glc2. (B) Close-up view rotated 45° showing the 2Fo-Fc electron density map (1.5 σ) of the maltoheptaose chain (green) and phosphate (orange) bound to SEX4. The density permitted modeling of six glucose moieties in the maltoheptaose chain (numbered from nonreducing to the reducing end) and clear assignment of glucan orientation. Two conformers were modeled for Glc1, differing in the orientation of the O6 group.

Table 1.

Data collection and refinement statistics

	Δ91 SEX4 (C193S); maltoheptaose and phosphate
Data collection
Beamline	APS 22-ID
Space group	P212121
Cell dimensions (a, b, c)	33.48, 77.89, 117.21
Unique reflections	36,096
Completeness, %	95.2 (91.0)
Resolution, Å	20.0–1.65 (1.71–1.65)
Rmerge %	8.0 (43.6)
Redundancy	3.3 (2.8)
I/σI	11.0 (2.3)
Refinement
Resolution limits, Å	19.43–1.65
No. of reflections/no. to compute Rfree	34,257/1,807
R (Rfree)	17.9 (21.8)
No. of protein residues	293
No. of solvent molecules	355
No. of ligands	2
B factors
Protein	17.3
Ligand/ion	18.9 (maltoheptaose)
	9.1 (phosphate)
Water	27.5
rmsd
Bond lengths, Å	0.009
Bond angles, °	1.52

Values in parentheses are for the highest resolution shell.

Maltoheptaose at the SEX4 CBM.

The SEX4 CBM interacts with the maltoheptaose chain moieties Glc4–6 (Fig. 2A). The central platform for this interaction is a dual-tryptophan motif formed from W278 and W314, which combines with H330 to interact with both faces of the glucan chain. In addition, N332 and K307 are positioned behind maltoheptaose and form hydrogen-bonding interactions with the O3 groups of Glc5 and Glc6, respectively. These five residues are highly conserved among SEX4 orthologs (Fig. S1) and form a concerted glucan-interacting interface.

Fig. 2.

Interaction of maltoheptaose with the SEX4 CBM. (A) Close-up of Glc4–6 (green) bound to the SEX4 CBM (pink). CBM residues (yellow) interact with Glc5 and Glc6 to form a binding interface. (B) Specific activity of CBM mutants against Arabidopsis starch. Error bars represent the ±SD of six replicates. Inactive mutant SEX4-C198S (SEX4 C/S) was used as a negative control. Statistical comparison of wild-type and mutant activity demonstrates significant differences, P < 0.001, between all constructs. (C) Results of the cosedimentation assay of SEX4 and CBM mutants with amylopectin. Amylopectin-bound proteins are found in the pellet (P) and unbound proteins are found in the soluble (S) fraction.

The SEX4 CBM belongs to the CBM48 family and the dual-tryptophan platform (W278/W314) and K307 represent a conserved functional motif in CBM48 and the related CBM20 family (42, 43). The most similar CBM to that of SEX4 is found in the AMP-activated protein kinase β-subunit (AMPK-β) (Protein Data Bank ID code 1Z0N), which has a root-mean-square deviation (rmsd) of 1.4 Å compared with the SEX4 CBM (44). AMPK-β contains all of the SEX4 CBM glucan-interacting residues except for a threonine where H330 is located in SEX4. A comparison between the maltoheptaose-bound and ligand-free SEX4 structures reveals that H330 undergoes a conformational shift upon glucan binding, bringing H330 directly in line with Glc6 and W314 (Fig. S2A) (38). This glucan-bound configuration is ideally structured for positioning a linear glucan chain in the SEX4 CBM-binding interface.

To determine the contribution of the CBM to overall SEX4 activity, we mutated the aforementioned CBM residues to alanine and tested the mutants’ ability to dephosphorylate native Arabidopsis starch, the endogenous substrate of SEX4 (Fig. 2B). Alanine point mutations of W278, K307, W314, H330, and N332 resulted in a decrease of total phosphatase activity ranging from 66% to 97%. The largest decrease resulted from the mutation of W278, which is located closest to the interface between the CBM and DSP domains of SEX4. All CBM mutants had generic para-nitrophenyl phosphate (pNPP) dephosphorylation levels comparable to wild type, indicating that decreases in glucan phosphatase activity were not due to aberrant folding (Fig. S3). Because CBMs typically engage substrates, these results suggest that a loss of starch phosphatase activity may indicate a decrease of starch binding. To test this, we incubated SEX4 proteins with amylopectin coupled to Con A Sepharose (GE Healthcare), washed the beads, and used Western analysis to determine if the protein was bound to amylopectin in the pellet or remained in the supernatant. We found that mutation of CBM residues resulted in a dramatic decrease in amylopectin binding (Fig. 2C). Together, these data demonstrate that the SEX4 CBM is essential for glucan binding and, consequentially, dephosphorylation of starch.

Maltoheptaose and Phosphate at the SEX4 DSP.

The binding site at the SEX4 CBM interface is continuous with a corresponding interface in the DSP that guides Glc1–4 of the maltoheptaose chain directly over the catalytic site (Fig. 3A). The maltoheptaose chain has a curved configuration at the DSP active site. The concave surface of the maltoheptaose chain interacts with F167, which was previously shown to be important for glucan phosphatase activity (38). The convex surface interacts with K237, F235, Y90, F140, and Y139. All of these residues are located in DSP subdomains whose variability among DSP family members corresponds with the specific substrate requirements of each particular phosphatase (45). Y90 is located in the Recognition Domain (residues 90–98), F140 and Y139 are in the variable (V) loop (131–157), F235 and K237 are located in the R motif (230–249), and F167 is located in the D loop (162–168) (Fig. S4). A comparison of the glucan-bound DSP with the non–glucan-bound SEX4 structure reveals that residues F167, F235, Y139, and K237 undergo a conformational shift upon glucan binding to engage the glucan chain (Fig. S2B). Based on these data, we hypothesized that the DSP active site is also essential for dephosphorylation of starch glucan chains.

Fig. 3.

Interaction of maltoheptaose and phosphate at the SEX4 DSP active site. (A) Close-up of maltoheptaose (green), phosphate (orange), and the DSP (blue) active site (red). Side chains of interacting residues are colored yellow. (B) Specific activity of DSP mutants against Arabidopsis starch. Error bars represent the ±SD of six replicates. Statistical comparison of wild-type and mutant activity demonstrates significant differences, P < 0.005, between all constructs. (C) Results of the cosedimentation assay of SEX4 and DSP mutants with amylopectin. Amylopectin-bound proteins are found in the pellet (P) and unbound proteins are found in the soluble (S) fraction. (D) Fo-Fc omit electron density map (2.5 σ) of maltoheptaose and phosphate shows clear glucan orientation of the O6 group pointed toward the catalytic site (red). O6 and O3 groups of the Glc2 moiety are labeled. Catalytic triad residues D166, R204, and S(C)198 are depicted in yellow.

To test this hypothesis, we generated alanine mutations of the identified DSP residues and determined the mutants’ ability to dephosphorylate Arabidopsis starch granules (Fig. 3B). Mutation of Y90, Y139, F140, F235, and K237 to alanine resulted in a decrease of total starch dephosphorylation ranging from 10% to 80%. Each mutant had generic pNPP dephosphorylation levels comparable to wild type, indicating that decreases were not due to aberrant folding (Fig. S3). Interestingly, the average decrease in SEX4 starch dephosphorylation activity upon DSP mutation (38%) was lower than the average decrease upon CBM mutation (89%). Furthermore, we found that the DSP mutants maintained near-wild type-binding to amylopectin (Fig. 3C). These data were surprising, given that the DSP has a larger relative contact area with the phosphoglucan substrate (60%) than the CBM (40%). However, the CBM is in contact with only the glucan whereas the DSP is in contact with both phosphate and glucan. We hypothesized that the CBM functions in engaging glucan chains while the DSP integrates and positions a phosphoglucan into the catalytic site, thereby dictating C6-specific activity.

Indeed the maltoheptaose chain within the DSP domain of the SEX4 structure is clearly positioned in a C6-specific orientation (Fig. 3D). The O6 group of Glc2 interacts with the phosphate in the catalytic site at a distance of 2.6 Å, compared with 7.1 Å for the O3 group. In addition, the glucose moieties upstream and downstream of Glc2 are also oriented with the C6 position toward the catalytic site. Interestingly, the CBM mutants H330A and K307A, which dramatically decreased overall glucan phosphatase activity (Fig. 2B), still maintained a C6-position site specificity that is nearly identical to wild-type SEX4 (Fig. S5). These data support the hypothesis that the DSP functions to control phosphoglucan orientation, i.e., site specificity.

Structural Basis of SEX4 C6 Specificity.

To determine the structural basis of the C6-specific orientation of maltoheptaose at the DSP, we examined the residues that form the surface of the SEX4 active site (Fig. 4A) and compared them to LSF2, which is a C3-specific glucan phosphatase (Fig. 4B) (26). Although the overall active sites of SEX4 and LSF2 are globally similar, we identified specific conserved differences within the active sites. In SEX4, F235 in the R motif and F140 in the V loop that interact with Glc3 and Glc1 at a distance of ∼3.8 Å. In contrast, LSF2 contains G230 in its R motif and W136 in its V loop at the same positions as SEX4 F235 and F140, respectively. In LSF2, G230 and W136 form hydrogen bonds with the glucose moieties upstream and downstream of the moiety at the catalytic site, whereas SEX4 uses van der Waals interactions at these positions. Furthermore, analysis of the active site surface shows that SEX4 contains a distinct ridge at the R motif, whereas LSF2 contains a groove at the same position (Fig. 4C). The ridge in SEX4 is created by the β-carbon of F235, and the groove in LSF2 results from the absence of a side chain at G230. We hypothesized that F235 and F140 in SEX4 combine to form a distinctive active site topology that positions the phosphoglucan chain in a C6-specific orientation at the site of catalysis.

Fig. 4.

Structural basis for C6 specificity in SEX4. (A) Active site of SEX4 consisting of the R motif, PTP loop, and V loop. Glc2 of the maltoheptaose chain (green) is oriented with the O6 position toward the phosphate (orange) in the catalytic site. R-motif residues F235 and V-loop residue F140 interact with Glc3 and Glc1, respectively. A cross-section of the active site surface (red lines) is overlaid onto the equivalent positions in the model. (B) Equivalent active site of glucan phosphatase LSF2 (40). Glc3 of the maltohexaose chain (green) is oriented with the O3 position toward the phosphate (orange) in the catalytic site. R-motif residue G230 and V-loop residue W136 interact with Glc2 and Glc4, respectively. A cross-section of the active site surface (purple lines) is overlaid onto the equivalent positions in the model. (C) Superimposed cross-sections of the active site surface from SEX4 (red) and LSF2 (purple). Positions of F235 and G230 (F and G) and F140 and W136 (F and W) in SEX4 and LSF2, respectively, are denoted. (D) Relative specific activity of SEX4 active-site mutants at the C6 (blue) and C3 positions (yellow) of Arabidopsis starch granules represented as the percentage of total position dephosphorylation per minute per microgram of protein. Error bars represent the ±SD of six replicates. Statistical comparison of wild-type and mutant C6 and C3 activity demonstrates significant differences, P < 0.001, between all constructs. (E) Representation of data from D as a percentage of C3-dephosphorylation relative to total dephosphorylation.

To test this hypothesis, we mutated F235 and F140 in SEX4 to the corresponding residues found in LSF2. If these residues influence substrate specificity in SEX4, then their mutation should result in a shift from preferential C6-position dephosphorylation to preferential C3-position dephosphorylation. We generated single-point mutants (SEX4-F140W and SEX4-F235G) and a double mutant (SEX4-F140W/F235G) and determined the relative ability of each mutant to dephosphorylate the C6 and C3 position of Arabidopsis starch granules (Fig. 4 D and E). Wild-type SEX4 dephosphorylates the C3 position of starch glucose at a rate of 29% of total dephosphorylation. Strikingly, both the F140W and F235G mutations increased the rate of C3 dephosphorylation to 51% of total dephosphorylation, effectively removing SEX4 C6 specificity. Even more remarkably, the F140W/F235G double mutant increased the ratio of C3 dephosphorylation to 77%, fully reversing the substrate preference of SEX4 from the C6 to the C3 position. These results clearly support our hypothesis and indicate that F235 and F140 in the SEX4 DSP active site are responsible for preferential C6 dephosphorylation of starch granules. Moreover, the ability to reverse site-specific activity via DSP mutagenesis provides valuable insights into the basis of substrate specificity for the glucan phosphatase family.

Discussion

Reversible starch phosphorylation is the central regulatory event governing the transition from starch synthesis to starch breakdown in plant cells. SEX4 is critical to starch catabolism and these data reveal the structural mechanism of its activity. Our data demonstrate the basis for the coordinated function of the SEX4 domains, with glucan engagement achieved via the CBM and phosphoglucan positioning and site specificity via the DSP domain. We further demonstrate that two residues in the active site control the specificity of SEX4. These results establish the structural basis for the specific activity of glucan phosphatases.

Within the SEX4 glucan-binding interface, the CBM interacts with a nonphosphorylated glucan and the DSP integrates a phosphoglucan into the DSP active site. Although the DSP clearly positions the phosphoglucan, our starch-binding and dephosphorylation data demonstrate that the DSP plays only a minor role in glucan binding and demonstrates that the CBM is required to engage the glucan. DSPs function via nucleophilic attack of the phosphorus atom by the catalytic cysteine followed by formation and then hydrolysis of a phosphoenzyme intermediate (28, 31, 32). The integration of the phosphoglucan into the catalytic site by the DSP, with minimal contribution to overall glucan binding, is optimal for both formation of the specific phosphoenzyme intermediate and disengagement of the product. Thus, the SEX4 mechanism is ideal for enhancing substrate/product turnover.

Despite being composed of only α-linked glucose, starch is a complex substrate containing two glucose polymers, α-1,6-branch points, variable chain lengths, and a helical secondary structure. Thus, SEX4 must access phosphoglucans within this heterogenous landscape. Although phosphate positioning within glucan chains of amylopectin is currently unknown, our structure suggests that multiple C6-phosphate modifications could be accommodated at Glc1, Glc2, Glc5, and Glc6. Additionally, the structure indicates that an α-1,6-branch could be accommodated at both glucan termini and at Glc5. Glucan chains within amylopectin also possess an inherent torsion as they wrap into double-helical structures (4, 6). It is of note that the bound glucan chain possesses both curvature and torsion, with the C6 hydroxyls of Glc5–6 in the CBM pointed toward the solvent and away from the protein, whereas they are pointed toward the protein in Glc1–3 at the DSP (Fig. S6). Thus, SEX4 accommodates a helical glucan and may influence the geometry of the chain during binding.

The structure reveals that SEX4 accommodates six glucan monomers within its extended active site, with Glc2 positioned over the active site. Because both ends of the bound glucan chain are directed toward the solvent, it is possible that SEX4 can accommodate register shifts that would facilitate processivity. Given the domain-specific contributions to glucan binding and positioning, it is possible that SEX4 is able to diffuse along the glucan chain via the CBM and then integrate a phosphoglucan into the catalytic site. In Arabidopsis leaf starch, the frequency of glucosyl phosphorylation is ∼1 in 2,000 (18). Although phosphorylation is likely higher during starch degradation, the challenges of locating a phosphorylated glucosyl residue may require this more coordinated, and possibly processive, phosphoglucan engagement with the DSP–CBM operating as an integrated unit.

The DSP active site of SEX4 comprises DSP subdomains that have been adapted to dephosphorylate starch via coordination of aromatic and hydrophilic residues that form a glucan-binding platform. An aromatic platform was also found in LSF2, which is 45% identical to the SEX4 DSP (26). Comparative analysis between SEX4 and LSF2 reveals common features, as well as unique specificity-determining regions, in their active sites. Both enzymes contain broad and shallow active sites that engage three glucose moieties of an α-1,4-glycosidic–linked chain, but the active site topology of each enzyme is finely tuned to direct glucan orientation. The LSF2 active site makes a substantial contribution to glucan binding, unlike the SEX4 active site (26). Thus, each glucan phosphatase contains common and specific elements that allow it to engage specific phosphoglucan substrates.

The establishment of glucan-interacting motifs identified in SEX4 and LSF2 provides a basis for comparison with the noncatalytic DSP LSF1 and the human glycogen phosphatase laforin. In planta studies have demonstrated that LSF1 functions in starch metabolism, although its precise role is unknown (46). LSF1 is similar in domain architecture with SEX4, however the failure of LSF1 to dephosphorylate glucan substrates may stem from the absence of glucan-interacting platforms found in LSF2 and SEX4. Laforin dephosphorylates glycogen and possesses a CBM and DSP similar to SEX4, but the domains are in the opposite order (24, 33, 34). Mutations in the gene that encodes laforin result in Lafora disease, in which cellular glycogen is hyperphosphorylated and forms amylopectin-like inclusions (47). Initial analyses suggest that some Lafora disease patient mutations are within putative glucan-interacting platforms similar to those identified in SEX4. These aromatic- and hydrophilic-enriched, glucan-binding platforms represent a canonical theme in plant glucan phosphatases that may also be found in related enzymes.

Reversible phosphorylation is critical for starch breakdown in plants, and therefore represents a potential tool to modify the physical properties of starch or increase its yield in industrial and agricultural settings (12, 41). The myriad food and nonfood starch-based industrial products require enzymatic and chemical modification of starch to generate necessary starch-based feedstocks (48–50). Phosphorylation is the only known natural modification of starch, and has direct influences on starch hydration, crystallinity, freeze–thaw stability, viscosity, and transparency that are central to various commercial applications (39–41). Furthermore, it is clear that C6 and C3 phosphorylation of starch influence its properties differently, as studies show that C3 phosphate has a more direct effect on starch granule solubilization (14). The present study illustrates that the C6 and C3 specificity of SEX4 can be altered via the engineering of a discrete number of DSP residues and reversed site-specific activity in SEX4 would certainly influence the pattern of starch metabolism in planta. Engineered SEX4 could provide a means to generate designer starches with tailored patterns of phosphorylation and physical characteristics useful in industrial settings. Recently, GWD-silenced wheat plants were shown to have a significant increase in starch yield (51), and silencing or engineering SEX4 activity in crop plants may produce similarly favorable results. Elucidation of the structural basis of SEX4 activity provides valuable insights necessary for its application in biotechnology.

Materials and Methods

Cloning, expression and purification of A. thaliana Δ89-SEX4 wild type and mutants (38), as well as recombinant GWD and PWD (19, 21), were performed as previously described. A. thaliana Δ89-SEX4 C198S (inactive mutant) protein used for crystallization was preincubated with 25 mM maltoheptaose (Sigma-Aldrich). The crystallization screen trials crystals were set up via hanging drop vapor diffusion using a Mosquito liquid-handling robot (TTPLabtech) using a 200-nL drop with a 1:1 ratio of condition and 12 mg/mL protein. Ligand-bound crystals were obtained in 0.2 M MgCl₂ and 20% (wt/vol) PEG 3350. Crystals were briefly soaked in mother liquor with 20% (vol/vol) glycerol and flash-frozen. A single crystal was used for data collection and structural determination. Data were collected on the 22-ID beamline of the Southeast Regional Collaborative Access Team (Advanced Photon Source, Argonne National Laboratory, Argonne, IL) (Table 1) at 120 K at a wavelength of 1.00 Å. There was one molecule in the asymmetric unit and the structure was determined using molecular replacement with the B chain of the previously determined SEX4 structure as the search model (38). Phosphatase assays using pNPP (24, 33, 35), phosphate-free Arabidopsis sex1-3 starch (16, 22, 26), and the glucan-binding assay (52) were performed as previously described. Additional details regarding procedures and data analysis are provided in SI Materials and Methods.