The Laforin-Like Dual-Specificity Phosphatase SEX4 from Arabidopsis Hydrolyzes Both C6- and C3-Phosphate Esters Introduced by Starch-Related Dikinases and Thereby Affects Phase Transition of α-Glucans

Abstract

The biochemical function of the Laforin-like dual-specific phosphatase AtSEX4 (EC 3.1.3.48) has been studied. Crystalline maltodextrins representing the A-or the B-type allomorph were prephosphorylated using recombinant glucan, water dikinase (StGWD) or the successive action of both plastidial dikinases (StGWD and AtPWD). AtSEX4 hydrolyzed carbon 6-phosphate esters from both the prephosphorylated A-and B-type allomorphs and the kinetic constants are similar. The phosphatase also acted on prelabeled carbon-3 esters from both crystalline maltodextrins. Similarly, native starch granules prelabeled in either the carbon-6 or carbon-3 position were also dephosphorylated by AtSEX4. The phosphatase did also hydrolyze phosphate esters of both prephosphorylated maltodextrins when the (phospho)glucans had been solubilized by heat treatment. Submillimolar concentrations of nonphosphorylated maltodextrins inhibited AtSEX4 provided they possessed a minimum of length and had been solubilized. As opposed to the soluble phosphomaltodextrins, the AtSEX4-mediated dephosphorylation of the insoluble substrates was incomplete and at least 50% of the phosphate esters were retained in the pelletable (phospho)glucans. The partial dephosphorylation of the insoluble glucans also strongly reduced the release of nonphosphorylated chains into solution. Presumably, this effect reflects fast structural changes that following dephosphorylation occur near the surface of the maltodextrin particles. A model is proposed defining distinct stages within the phosphorylation/ dephosphorylation-dependent transition of a-glucans from the insoluble to the soluble state.The metabolism of starch, the most prominent storage carbohydrate in plants, is assumed to require approximately 30 to 40 distinct (iso)enzymes (Deschamps et al., 2008), but, presumably, the list of the starch-related proteins is not yet complete. Several novel proteins (and protein functions) essential for the normal starch metabolism have recently been identified among which are two a-glucan phosphorylating dikinases. One dikinase (glucan, water dikinase [GWD], EC 2.7.9.4) utilizes ATP as dual phosphate donor and esterifies the C6 position of amylopectin-related glucosyl residues, whereas the other dikinase (phosphoglucan, water dikinase [PWD], EC 2.7.9.5) selectively transfers the b-phosphate group from ATP to the C3 position of glucosyl residues (Ritte et al., 2006).Two other previously unknown starch-related enzymes were designated as SEX4 protein (EC 3

Structural basis for the glucan phosphatase activity of Starch Excess4

“…SEX4 dephosphorylates both the starch granule surface as well as soluble phosphoglucans (16). In addition, SEX4 can dephosphorylate both the C3 and C6 position of glucose (22). Therefore, SEX4 possesses a broad substrate specificity and must accommodate both different glucans (i.e., soluble and crystalline) as well as differently positioned phosphates (i.e., C3 versus C6).…”

“…In addition to dephosphorylating amylopectin (18), recombinant SEX4 also dephosphorylates crystalline maltodextrins (22), starch granules isolated from A. thaliana leaves (16), and phospho-oligosaccharides (16). While C6 and C3 phosphorylation is carried out by two dikinases, SEX4 is able to dephosphorylate both C6 and C3 positions with similar kinetics (22). Thus, it appears that SEX4 possesses a broader substrate specificity than the kinases, in terms of phosphate position, length of glucan, and solubility of the glucan.…”

“…Mutations in the SEX4 gene result in substantial accumulations of starch in A. thaliana leaves due to decreased rates of degradation and the accumulation of soluble phospho-oligosaccharides (16,(19)(20)(21). In addition to dephosphorylating amylopectin (18), recombinant SEX4 also dephosphorylates crystalline maltodextrins (22), starch granules isolated from A. thaliana leaves (16), and phospho-oligosaccharides (16). While C6 and C3 phosphorylation is carried out by two dikinases, SEX4 is able to dephosphorylate both C6 and C3 positions with similar kinetics (22).…”

Metabolism and Growth in Arabidopsis Depend on the Daytime Temperature but Are Temperature-Compensated against Cool Nights

“…A cycle of glucan phosphorylation (Kötting et al, 2009;Comparot-Moss et al, 2010) and dephosphorylation (Zeeman et al, 2007 is required for the initial attack on the starch granule, possibly by facilitating a phase transition that makes the surface of the crystalline starch granule accessible to attack by b-amylases (Hejazi et al, 2010). Low night temperatures led to an increase in transcripts for proteins involved in starch degradation, in particular in this cycle.…”

“…There was also a widespread increase in transcripts for starch degradation, including five that encode proteins involved in a cycle of glucan phosphorylation and dephosphorylation that is the first step in starch degradation (GWD1, GWD2, GWD3, SEX4, and PTPKIS2; Figure 5; Kötting et al, 2009;Comparot-Moss et al, 2010;Hejazi et al, 2010;Zeeman et al, 2010) and two plastidial b-amylases (BAM2 and BAM3; Fulton et al, 2008) that are thought to be involved in the initial attack on the starch granule. As previously seen (Smith et al, 2004;Usadel et al, 2008a), most transcripts for starch degradation (with the exception of the BAM family) showed large diurnal changes, with very low levels at the EN and high levels at the end of the light period.…”