Surface binding sites in amylase have distinct roles in recognition of starch structure motifs and degradation
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Surface binding sites in amylase have distinct roles in recognition of starch structure motifs and degradation. / Cockburn, Darrell; Nielsen, Morten Munch; Christiansen, Camilla; Andersen, Joakim Mark; Rannes, Julie Bille; Blennow, Andreas; Svensson, Birte.
In: International Journal of Biological Macromolecules, Vol. 75, 2015, p. 338-345.Research output: Contribution to journal › Journal article › Research › peer-review
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TY - JOUR
T1 - Surface binding sites in amylase have distinct roles in recognition of starch structure motifs and degradation
AU - Cockburn, Darrell
AU - Nielsen, Morten Munch
AU - Christiansen, Camilla
AU - Andersen, Joakim Mark
AU - Rannes, Julie Bille
AU - Blennow, Andreas
AU - Svensson, Birte
N1 - Copyright © 2015 Elsevier B.V. All rights reserved.
PY - 2015
Y1 - 2015
N2 - Carbohydrate converting enzymes often possess extra substrate binding regions that enhance their activity. These can be found either on separate domains termed carbohydrate binding modules or as so-called surface binding sites (SBSs) situated on the catalytic domain. SBSs are common in starch degrading enzymes and critically important for their function. The affinity towards a variety of starch granules as well as soluble poly- and oligosaccharides of barley α-amylase 1 (AMY1) wild-type and mutants of two SBSs (SBS1 and SBS2) was investigated using Langmuir binding analysis, confocal laser scanning microscopy, affinity gel electrophoresis and surface plasmon resonance to unravel functional roles of the SBSs. SBS1 was critical for binding to different starch types as Kd increased by 7-62-fold or was not measurable upon mutation. By contrast SBS2 was particularly important for binding to soluble polysaccharides and oligosaccharides with α-1,6 linkages, suggesting that branch points are key structural elements in recognition by SBS2. Mutation at both SBS1 and SBS2 eliminated binding to all starch granule types tested. Taken together, the findings indicate that the two SBSs act in concert to localize AMY1 to the starch granule surface and that SBS2 works synergistically with the active site in the degradation of amylopectin.
AB - Carbohydrate converting enzymes often possess extra substrate binding regions that enhance their activity. These can be found either on separate domains termed carbohydrate binding modules or as so-called surface binding sites (SBSs) situated on the catalytic domain. SBSs are common in starch degrading enzymes and critically important for their function. The affinity towards a variety of starch granules as well as soluble poly- and oligosaccharides of barley α-amylase 1 (AMY1) wild-type and mutants of two SBSs (SBS1 and SBS2) was investigated using Langmuir binding analysis, confocal laser scanning microscopy, affinity gel electrophoresis and surface plasmon resonance to unravel functional roles of the SBSs. SBS1 was critical for binding to different starch types as Kd increased by 7-62-fold or was not measurable upon mutation. By contrast SBS2 was particularly important for binding to soluble polysaccharides and oligosaccharides with α-1,6 linkages, suggesting that branch points are key structural elements in recognition by SBS2. Mutation at both SBS1 and SBS2 eliminated binding to all starch granule types tested. Taken together, the findings indicate that the two SBSs act in concert to localize AMY1 to the starch granule surface and that SBS2 works synergistically with the active site in the degradation of amylopectin.
U2 - 10.1016/j.ijbiomac.2015.01.054
DO - 10.1016/j.ijbiomac.2015.01.054
M3 - Journal article
C2 - 25661878
VL - 75
SP - 338
EP - 345
JO - International Journal of Biological Macromolecules
JF - International Journal of Biological Macromolecules
SN - 0141-8130
ER -
ID: 132427603