Structure of the Arabidopsis Glucan Phosphatase LIKE SEX FOUR2 Reveals a Unique Mechanism for Starch Dephosphorylation

Abstract

Starch is a water-insoluble, Glc-based biopolymer that is used for energy storage and is synthesized and degraded in a diurnal manner in plant leaves. Reversible phosphorylation is the only known natural starch modification and is required for starch degradation in planta. Critical to starch energy release is the activity of glucan phosphatases; however, the structural basis of dephosphorylation by glucan phosphatases is unknown. Here, we describe the structure of the Arabidopsis thaliana starch glucan phosphatase LIKE SEX FOUR2 (LSF2) both with and without phospho-glucan product bound at 2.3Å and 1.65Å, respectively. LSF2 binds maltohexaose-phosphate using an aromatic channel within an extended phosphatase active site and positions maltohexaose in a C3-specific orientation, which we show is critical for the specific glucan phosphatase activity of LSF2 toward native Arabidopsis starch. However, unlike other starch binding enzymes, LSF2 does not possess a carbohydrate binding module domain. Instead we identify two additional glucan binding sites located within the core LSF2 phosphatase domain. This structure is the first of a glucan-bound glucan phosphatase and provides new insights into the molecular basis of this agriculturally and industrially relevant enzyme family as well as the unique mechanism of LSF2 catalysis, substrate specificity, and interaction with starch granules.

Mechanistic Insights into Glucan Phosphatase Activity against Polyglucan Substrates

“…Site-specific glucan phosphate activity was determined by release from 33 P-labeled starch (15,27,28,57). C6-33 P-labeled starch was generated by purifying phosphate-free starch from the Arabidopsis sex1-3 mutant (63), phosphorylating the starch with 33 P at the C6-position by incubation with GWD and [␤-…”

“…All protein constructs were expressed and purified as previously described via the following protocol, except where noted (28,42,57). Protein was produced in BL21-Codon Plus Escherichia coli cells (Stratagene).…”

“…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.…”

“…Conversely, the DSP and CBM in laforin are spatially separated and engage glucan chains independently (42). LSF2, lacking a CBM, uses two non-catalytic secondary binding sites associated with its CT domain to interact with starch glucans at a distance of Ͼ20 Å from the DSP active site (57). These structural studies demonstrate that the glucan phosphatases are all capable of binding glucan chains via the DSP domain and ancillary glucanbinding domains, but they use distinct variations on this theme to achieve their respective glucan phosphatase functions within their own biological niches.…”

“…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).…”