PDBsum entry 4pyh

Hydrolase

PDB id: 4pyh

Contents

Protein chain

293 a.a.

Ligands

GLC-GLC-GLC-GLC-GLC-GLC

PO4

GOL

Metals

_MG

Waters ×354

PDB id:

4pyh

Name:

Hydrolase

Title:

Phospho-glucan bound structure of starch phosphatase starch excess4 reveals the mechanism for c6-specificty

Structure:

Phosphoglucan phosphatase dsp4, chloroplastic. Chain: a. Fragment: unp residues 90-379. Synonym: atptpkis1, dual specificity protein phosphatase 4, protein starch-excess 4, atsex4. Engineered: yes. Mutation: yes

Source:

Arabidopsis thaliana. Mouse-ear cress,thale-cress. Organism_taxid: 3702. Gene: dsp4, ptpkis1, sex4, at3g52180, f4f15.290. Expressed in: escherichia coli. Expression_system_taxid: 511693.

Resolution:

1.65Å R-factor: 0.179 R-free: 0.218

Authors:

D.A.Meekins,C.W.Vander Kooi

Key ref:

D.A.Meekins et al. (2014). Phosphoglucan-bound structure of starch phosphatase Starch Excess4 reveals the mechanism for C6 specificity. Proc Natl Acad Sci U S A, 111, 7272-7277. PubMed id: 24799671 DOI: 10.1073/pnas.1400757111

Date:

27-Mar-14 Release date: 23-Apr-14

Protein chain

Q9FEB5  (DSP4_ARATH) -  Phosphoglucan phosphatase DSP4, chloroplastic from Arabidopsis thaliana

Seq: 379 a.a.

Struc: 293 a.a.*

* PDB and UniProt seqs differ at 5 residue positions (black crosses)

Enzyme reactions

• Enzyme class: E.C.3.1.3.- - ?????

DOI no: 10.1073/pnas.1400757111

Proc Natl Acad Sci U S A 111:7272-7277 (2014)

PubMed id: 24799671

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

D.A.Meekins, M.Raththagala, S.Husodo, C.J.White, H.F.Guo, O.Kötting, C.W.Vander Kooi, M.S.Gentry.

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.