title: Starch-binding domains in the CBM45 family--low-affinity domains from glucan, water dikinase and α-amylase involved in plastidial starch metabolism.
abstract: Starch-binding domains are noncatalytic carbohydrate-binding modules that mediate binding to granular starch. The starch-binding domains from the carbohydrate-binding module family 45 (CBM45, http://www.cazy.org) are found as N-terminal tandem repeats in a small number of enzymes, primarily from photosynthesizing organisms. Isolated domains from representatives of each of the two classes of enzyme carrying CBM45-type domains, the Solanum tuberosumα-glucan, water dikinase and the Arabidopsis thaliana plastidial α-amylase 3, were expressed as recombinant proteins and characterized. Differential scanning calorimetry was used to verify the conformational integrity of an isolated CBM45 domain, revealing a surprisingly high thermal stability (T(m) of 84.8 °C). The functionality of CBM45 was demonstrated in planta by yellow/green fluorescent protein fusions and transient expression in tobacco leaves. Affinities for starch and soluble cyclodextrin starch mimics were measured by adsorption assays, surface plasmon resonance and isothermal titration calorimetry analyses. The data indicate that CBM45 binds with an affinity of about two orders of magnitude lower than the classical starch-binding domains from extracellular microbial amylolytic enzymes. This suggests that low-affinity starch-binding domains are a recurring feature in plastidial starch metabolism, and supports the hypothesis that reversible binding, effectuated through low-affinity interaction with starch granules, facilitates dynamic regulation of enzyme activities and, hence, of starch metabolism.
authors.affiliation: Enzyme and Protein Chemistry, Department of Systems Biology, Technical University of Denmark, Kongens Lyngby, Denmark.
authors.forename: Mikkel A
authors.initials: MA
authors.lastname: Glaring
authors.forename: Martin J
authors.initials: MJ
authors.lastname: Baumann
authors.forename: Maher
authors.initials: M
authors.lastname: Abou Hachem
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Nakai
authors.forename: Natsuko
authors.initials: N
authors.lastname: Nakai
authors.forename: Diana
authors.initials: D
authors.lastname: Santelia
authors.forename: Bent W
authors.initials: BW
authors.lastname: Sigurskjold
authors.forename: Samuel C
authors.initials: SC
authors.lastname: Zeeman
authors.forename: Andreas
authors.initials: A
authors.lastname: Blennow
authors.forename: Birte
authors.initials: B
authors.lastname: Svensson
mesh_terms: D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000737:chemistry / Q000166:cytology / Q000378:metabolism; D002151:Calorimetry; D005936:Glucans / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018087:Plastids / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D011198:Solanum tuberosum / Q000737:chemistry / Q000166:cytology / Q000378:metabolism; D013213:Starch / Q000378:metabolism*; D020349:Surface Plasmon Resonance; D014026:Nicotiana / Q000737:chemistry / Q000166:cytology / Q000378:metabolism; D000516:alpha-Amylases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D005936:Glucans; D010940:Plant Proteins; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins; C055213:saccharide-binding proteins; D013213:Starch; D000516:alpha-Amylases