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Discovery, characterization, and structural analyses of glucan phosphatases from plants to humans
Author(s) -
Gentry Matthew S.,
Meekins David A.,
Taylor Adam O.,
Santelia Diana,
Kotting Oliver,
Zeeman Samuel C.,
Vander Kooi Craig W.
Publication year - 2012
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.26.1_supplement.766.3
Subject(s) - dephosphorylation , phosphatase , biochemistry , phosphorylation , starch , glucan , dual specificity phosphatase , chemistry , phosphate
We initially demonstrated that the human phosphatase laforin and plant phosphatase S tarch EX cess4 (SEX4) are glucan phosphatases. Mutations in the gene encoding laforin cause glycogen to become hyperphosphorylated, resulting in starch‐like glucans called Lafora bodies (LBs) and the fatal neurodegenerative disorder Lafora's disease. Plant starch contains covalently bound phosphate at the C6‐ and C3‐position of glucosyl residues. Reversible phosphorylation of starch outer glucans renders these glucans accessible to hydrolyzing enzymes. In the absence of starch phosphorylation or dephosphorylation, plants accumulate excessively large starch granules that they are unable to breakdown. SEX4 activity is a necessary step in the starch degradation cycle of glucan phosphorylation, hydrolysis, and dephosphorylation. We utilized X‐ray crystallography to determine the structure of SEX4 and this structure assisted in the identification of L ike S ex F our2 (LSF2). Unlike SEX4, LSF2 lacks a carbohydrate‐binding module yet LSF2 binds starch. We found that SEX4 releases phosphate from the C3‐ and C6‐position, but prefers C6. Alternatively, LSF2 hydrolyzes phosphate from the C3‐position and plants lacking LSF2 have elevated C3‐bound phosphate. Lastly, we determined the crystal structure of LSF2 and utilize site‐directed mutagenesis with in vitro assays to define the structural elements of glucan phosphatases.