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?-glucan, water dikinase (GWD): a plastidic enzyme with redox-regulated and coordinated catalytic activity and binding affinity

?-glucan, water dikinase (GWD): a plastidic enzyme with redox-regulated and coordinated catalytic activity and binding affinity
?-glucan, water dikinase (GWD): a plastidic enzyme with redox-regulated and coordinated catalytic activity and binding affinity
The recently discovered potato tuber (Solanum tuberosum) {alpha}-glucan, water dikinase (GWD) (formerly known as R1) catalyzes the phosphorylation of starch by a dikinase-type reaction mechanism in which the {beta}-phosphate of ATP is transferred to either the C-6 or the C-3 position of the glucosyl residue of starch. In the present study, we found that the GWD enzyme is inactive in the oxidized form, which is accompanied by the formation of a specific intramolecular disulfide bond as determined by disulfide-linked peptide mapping. The regulatory properties of this disulfide linkage were confirmed by site-directed mutagenesis studies. Both reduced thioredoxin (Trx) f and Trx m from spinach leaves reduced and activated oxidized GWD at very low concentrations, with Trx f being the more efficient, yielding an S0.5 value of 0.4 µM. Interestingly, GWD displays a reversible and selective binding to starch granules depending on the illumination state of the plant. Here we show that starch granule-bound GWD isolated from dark-adapted plants exists in the inactive, oxidized form, which is capable of reactivation upon treatment with reduced Trx. Furthermore, the soluble form of GWD was found in its fully reduced state, providing evidence of a Trx-controlled regulation mechanism linking enzymatic activity and specific binding affinities of a protein to an intracellular surface. The regulatory site sequence, CFATC, of potato GWD is conserved in chloroplast-targeted GWDs from other species, suggesting an overall redox regulation of the GWD enzyme.
starch, redox regulation, thioredoxin
0027-8424
1785-1790
Mikkelsen, René
0c85146a-6d1e-41f8-8845-f6a08d912e58
Mutenda, Kudzai E.
e7cdd4a2-1e5c-44a4-8340-058a5161a4ba
Mant, Alexandra
63319e45-deeb-45ad-a30d-e05b42052a0d
Schürmann, Peter
d82d13a8-ae6e-45a4-9516-5a615d517237
Blennow, Andreas
77de1c12-a00c-4b04-b639-c6e8892f390c
Mikkelsen, René
0c85146a-6d1e-41f8-8845-f6a08d912e58
Mutenda, Kudzai E.
e7cdd4a2-1e5c-44a4-8340-058a5161a4ba
Mant, Alexandra
63319e45-deeb-45ad-a30d-e05b42052a0d
Schürmann, Peter
d82d13a8-ae6e-45a4-9516-5a615d517237
Blennow, Andreas
77de1c12-a00c-4b04-b639-c6e8892f390c

Mikkelsen, René, Mutenda, Kudzai E., Mant, Alexandra, Schürmann, Peter and Blennow, Andreas (2005) ?-glucan, water dikinase (GWD): a plastidic enzyme with redox-regulated and coordinated catalytic activity and binding affinity. Proceedings of the National Academy of Sciences of the United States of America, 102 (5), 1785-1790. (doi:10.1073/pnas.0406674102).

Record type: Article

Abstract

The recently discovered potato tuber (Solanum tuberosum) {alpha}-glucan, water dikinase (GWD) (formerly known as R1) catalyzes the phosphorylation of starch by a dikinase-type reaction mechanism in which the {beta}-phosphate of ATP is transferred to either the C-6 or the C-3 position of the glucosyl residue of starch. In the present study, we found that the GWD enzyme is inactive in the oxidized form, which is accompanied by the formation of a specific intramolecular disulfide bond as determined by disulfide-linked peptide mapping. The regulatory properties of this disulfide linkage were confirmed by site-directed mutagenesis studies. Both reduced thioredoxin (Trx) f and Trx m from spinach leaves reduced and activated oxidized GWD at very low concentrations, with Trx f being the more efficient, yielding an S0.5 value of 0.4 µM. Interestingly, GWD displays a reversible and selective binding to starch granules depending on the illumination state of the plant. Here we show that starch granule-bound GWD isolated from dark-adapted plants exists in the inactive, oxidized form, which is capable of reactivation upon treatment with reduced Trx. Furthermore, the soluble form of GWD was found in its fully reduced state, providing evidence of a Trx-controlled regulation mechanism linking enzymatic activity and specific binding affinities of a protein to an intracellular surface. The regulatory site sequence, CFATC, of potato GWD is conserved in chloroplast-targeted GWDs from other species, suggesting an overall redox regulation of the GWD enzyme.

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More information

Published date: 2005
Keywords: starch, redox regulation, thioredoxin
Organisations: Cancer Sciences

Identifiers

Local EPrints ID: 26484
URI: http://eprints.soton.ac.uk/id/eprint/26484
ISSN: 0027-8424
PURE UUID: dce6f6eb-5d4e-4e73-b202-94f68133a1a7
ORCID for Alexandra Mant: ORCID iD orcid.org/0000-0001-7169-209X

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Date deposited: 12 Apr 2006
Last modified: 16 Mar 2024 03:40

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Contributors

Author: René Mikkelsen
Author: Kudzai E. Mutenda
Author: Alexandra Mant ORCID iD
Author: Peter Schürmann
Author: Andreas Blennow

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