Functional demonstrations of starch binding domains present in Ostreococcus tauri starch synthases isoforms.
BACKGROUND
Starch-binding domains are key modules present in several enzymes involved in polysaccharide metabolism. These non-catalytic modules have already been described as essential for starch-binding and the catalytic activity of starch synthase III from the higher plant Arabidopsis thaliana. In Ostreococcus tauri, a unicellular green alga of the Prasinophyceae family, there are three SSIII isoforms, known as Ostta SSIII-A, SSIII-B and SSIII-C.


RESULTS
In this work, using in silico and in vitro characterization techniques, we have demonstrated that Ostta SSIII-A, SSIII-B and SSIII-C contain two, three and no starch-binding domains, respectively. Additionally, our phylogenetic analysis has indicated that OsttaSSIII-B, presenting three N-terminal SBDs, is the isoform more closely related to higher plant SSIII. Furthermore, the sequence alignment and homology modeling data gathered showed that both the main 3-D structures of all the modeled domains obtained and the main amino acid residues implicated in starch binding are well conserved in O. tauri SSIII starch-binding domains. In addition, adsorption assays showed that OsttaSSIII-A D2 and SSIII-B D2 domains are the two that make the greatest contribution to amylose and amylopectin binding, while OsttaSSIII-B D1 is also important for starch binding.


CONCLUSIONS
The results presented here suggest that differences between OsttaSSIII-A and SSIII-B SBDs in the number of and binding of amino acid residues may produce differential affinities for each isoform to polysaccharides. Increasing the knowledge about SBDs may lead to their employment in biomedical and industrial applications.
Centro de Estudios Fotosintéticos y Bioquímicos (CEFOBI-CONICET), Universidad Nacional de Rosario, Suipacha 531, 2000, Rosario, Argentina. barchiesi@cefobi-conicet.gov.ar.
Julieta
J
Barchiesi
Centro de Estudios Fotosintéticos y Bioquímicos (CEFOBI-CONICET), Universidad Nacional de Rosario, Suipacha 531, 2000, Rosario, Argentina. hedin@cefobi-conicet.gov.ar.
Nicolás
N
Hedin
Centro de Estudios Fotosintéticos y Bioquímicos (CEFOBI-CONICET), Universidad Nacional de Rosario, Suipacha 531, 2000, Rosario, Argentina. gomezcasati@cefobi-conicet.gov.ar.
Diego F
DF
Gomez-Casati
Department of Chemistry and Biochemistry, Loyola University Chicago, 405 Flanner Hall, 1068 W Sheridan Road, Chicago, IL, 60660, USA. mballic@luc.edu.
Miguel A
MA
Ballicora
Centro de Estudios Fotosintéticos y Bioquímicos (CEFOBI-CONICET), Universidad Nacional de Rosario, Suipacha 531, 2000, Rosario, Argentina. busi@cefobi-conicet.gov.ar.
María V
MV
Busi
D020418:Algal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D000687:Amylopectin / Q000737:chemistry* / Q000378:metabolism; D000688:Amylose / Q000737:chemistry* / Q000378:metabolism; D017360:Arabidopsis / Q000737:chemistry / Q000201:enzymology / Q000235:genetics; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D000460:Chlorophyta / Q000737:chemistry* / Q000201:enzymology / Q000235:genetics; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005964:Glucosyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D011485:Protein Binding; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013214:Starch Synthase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D040681:Structural Homology, Protein
D020418:Algal Proteins; D029681:Arabidopsis Proteins; D020033:Protein Isoforms; D011994:Recombinant Proteins; D000688:Amylose; D000687:Amylopectin; D005964:Glucosyltransferases; C504530:starch synthase III, Arabidopsis; D013214:Starch Synthase