In Vitro Biochemical Characterization of All Barley Endosperm Starch Synthases
Starch is the main storage polysaccharide in cereals and the major source of calories in the human diet. It is synthesized by a panel of enzymes including five classes of starch synthases (SSs). While the overall starch synthase (SS) reaction is known, the functional differences between the five SS...
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doaj-31ce6c33df0e4006b27b8850758fc6b22020-11-24T23:49:23ZengFrontiers Media S.A.Frontiers in Plant Science1664-462X2016-01-01610.3389/fpls.2015.01265170196In Vitro Biochemical Characterization of All Barley Endosperm Starch SynthasesJose Antonio Cuesta-Seijo0Morten M. Nielsen1Christian eRuzanski2Christian eRuzanski3Katarzyna eKrucewicz4Sophie R. Beeren5Sophie R. Beeren6Maja G. Rydhal7Yayoi eYoshimura8Yayoi eYoshimura9Alexander eStriebeck10Mohammed S. Motawia11William G.T. Willats12Monica M. Palcic13Carlsberg LaboratoryCarlsberg LaboratoryCarlsberg LaboratoryNovo Nordisk A/SCarlsberg LaboratoryCarlsberg LaboratoryTechnical University of DenmarkUniversity of CopenhagenCarlsberg LaboratoryGunma University Tenjin-choCarlsberg LaboratoryUniversity of CopenhagenUniversity of CopenhagenUniversity of CopenhagenStarch is the main storage polysaccharide in cereals and the major source of calories in the human diet. It is synthesized by a panel of enzymes including five classes of starch synthases (SSs). While the overall starch synthase (SS) reaction is known, the functional differences between the five SS classes are poorly understood. Much of our knowledge comes from analyzing mutant plants with altered SS activities, but the resulting data are often difficult to interpret as a result of pleitropic effects, competition between enzymes, overlaps in enzyme activity and disruption of multi-enzyme complexes. Here we provide a detailed biochemical study of the activity of all five classes of SSs in barley endosperm. Each enzyme was produced recombinantly in E. coli and the properties and modes of action in vitro were studied in isolation from other SSs and other substrate modifying activities. Our results define the mode of action of each SS class in unprecedented detail; we analyze their substrate selection, temperature dependence and stability, substrate affinity and temporal abundance during barley development. Our results are at variance with some generally accepted ideas about starch biosynthesis and might lead to the reinterpretation of results obtained in planta. In particular, they indicate that granule bound SS is capable of processive action even in the absence of a starch matrix, that SSI has no elongation limit, and that SSIV, believed to be critical for the initiation of starch granules, has maltoligosaccharides and not polysaccharides as its preferred substrates.http://journal.frontiersin.org/Journal/10.3389/fpls.2015.01265/fullSubstrate SpecificityKineticsAffinitystabilitybarleybiochemical characterization |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Jose Antonio Cuesta-Seijo Morten M. Nielsen Christian eRuzanski Christian eRuzanski Katarzyna eKrucewicz Sophie R. Beeren Sophie R. Beeren Maja G. Rydhal Yayoi eYoshimura Yayoi eYoshimura Alexander eStriebeck Mohammed S. Motawia William G.T. Willats Monica M. Palcic |
spellingShingle |
Jose Antonio Cuesta-Seijo Morten M. Nielsen Christian eRuzanski Christian eRuzanski Katarzyna eKrucewicz Sophie R. Beeren Sophie R. Beeren Maja G. Rydhal Yayoi eYoshimura Yayoi eYoshimura Alexander eStriebeck Mohammed S. Motawia William G.T. Willats Monica M. Palcic In Vitro Biochemical Characterization of All Barley Endosperm Starch Synthases Frontiers in Plant Science Substrate Specificity Kinetics Affinity stability barley biochemical characterization |
author_facet |
Jose Antonio Cuesta-Seijo Morten M. Nielsen Christian eRuzanski Christian eRuzanski Katarzyna eKrucewicz Sophie R. Beeren Sophie R. Beeren Maja G. Rydhal Yayoi eYoshimura Yayoi eYoshimura Alexander eStriebeck Mohammed S. Motawia William G.T. Willats Monica M. Palcic |
author_sort |
Jose Antonio Cuesta-Seijo |
title |
In Vitro Biochemical Characterization of All Barley Endosperm Starch Synthases |
title_short |
In Vitro Biochemical Characterization of All Barley Endosperm Starch Synthases |
title_full |
In Vitro Biochemical Characterization of All Barley Endosperm Starch Synthases |
title_fullStr |
In Vitro Biochemical Characterization of All Barley Endosperm Starch Synthases |
title_full_unstemmed |
In Vitro Biochemical Characterization of All Barley Endosperm Starch Synthases |
title_sort |
in vitro biochemical characterization of all barley endosperm starch synthases |
publisher |
Frontiers Media S.A. |
series |
Frontiers in Plant Science |
issn |
1664-462X |
publishDate |
2016-01-01 |
description |
Starch is the main storage polysaccharide in cereals and the major source of calories in the human diet. It is synthesized by a panel of enzymes including five classes of starch synthases (SSs). While the overall starch synthase (SS) reaction is known, the functional differences between the five SS classes are poorly understood. Much of our knowledge comes from analyzing mutant plants with altered SS activities, but the resulting data are often difficult to interpret as a result of pleitropic effects, competition between enzymes, overlaps in enzyme activity and disruption of multi-enzyme complexes. Here we provide a detailed biochemical study of the activity of all five classes of SSs in barley endosperm. Each enzyme was produced recombinantly in E. coli and the properties and modes of action in vitro were studied in isolation from other SSs and other substrate modifying activities. Our results define the mode of action of each SS class in unprecedented detail; we analyze their substrate selection, temperature dependence and stability, substrate affinity and temporal abundance during barley development. Our results are at variance with some generally accepted ideas about starch biosynthesis and might lead to the reinterpretation of results obtained in planta. In particular, they indicate that granule bound SS is capable of processive action even in the absence of a starch matrix, that SSI has no elongation limit, and that SSIV, believed to be critical for the initiation of starch granules, has maltoligosaccharides and not polysaccharides as its preferred substrates. |
topic |
Substrate Specificity Kinetics Affinity stability barley biochemical characterization |
url |
http://journal.frontiersin.org/Journal/10.3389/fpls.2015.01265/full |
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