INVESTIGADORES
GOMEZ CASATI Diego Fabian
artículos
Título:
The starch binding capacity of the non-catalytic SBD2 region and the interaction between the N- and C-terminal domains are involved in the modulation of the activity of starch synthase III from Arabidopsis thaliana.
Autor/es:
WAYLLACE, N. Z.; VALDEZ, H.; UGALDE, R.; BUSI, M. V.; DIEGO FABIAN GOMEZ CASATI
Revista:
FEBS JOURNAL
Editorial:
WILEY-BLACKWELL PUBLISHING, INC
Referencias:
Lugar: Cambridge; Año: 2010 vol. 277 p. 428 - 440
ISSN:
1742-464X
Resumen:
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Starch synthase III from Arabidopsis thaliana contains an N-terminal
region, including three in-tandem starch-binding domains, followed by a
C-terminal catalytic domain. We have reported previously that starch-binding
domains may be involved in the regulation of starch synthase III function.
In this work, we analyzed the existence of protein interactions
between both domains using pull-down assays, far western blotting and
co-expression of the full and truncated starch-binding domains with the
catalytic domain. Pull-down assays and co-purification analysis showed
that the D(316344) and D(495535) regions in the D2 and D3 domains,
respectively, but not the individual starch-binding domains, are involved in
the interaction with the catalytic domain. We also determined that the residues
W366 and Y394 in the D2 domain are important in starch binding.
Moreover, the co-purified catalytic domain plus site-directed mutants of
the D123 protein lacking these aromatic residues showed that W366 was
key to the apparent affinity for the polysaccharide substrate of starch synthase
III, whereas either of these amino acid residues altered ADP-glucose
kinetics. In addition, the analysis of full-length and truncated proteins
showed an almost complete restoration of the apparent affinity for the substrates
and Vmax of starch synthase III. The results presented here suggest
that the interaction of the N-terminal starch-binding domains, particularly
the D(316344) and D(495535) regions, with the catalytic domains, as well
as the full integrity of the starch-binding capacity of the D2 domain, are
involved in the modulation of starch synthase III activity.