title: Surface binding sites in amylase have distinct roles in recognition of starch structure motifs and degradation.
abstract: 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.
authors: Enzyme and Protein Chemistry, Department of Systems Biology, Technical University of Denmark, Søltofts Plads, Bldg. 224, DK-2800 Kgs Lyngby, Denmark.
authors: Darrell
authors: D
authors: Cockburn
authors: Enzyme and Protein Chemistry, Department of Systems Biology, Technical University of Denmark, Søltofts Plads, Bldg. 224, DK-2800 Kgs Lyngby, Denmark.
authors: Morten M
authors: MM
authors: Nielsen
authors: Enzyme and Protein Chemistry, Department of Systems Biology, Technical University of Denmark, Søltofts Plads, Bldg. 224, DK-2800 Kgs Lyngby, Denmark; Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark.
authors: Camilla
authors: C
authors: Christiansen
authors: Enzyme and Protein Chemistry, Department of Systems Biology, Technical University of Denmark, Søltofts Plads, Bldg. 224, DK-2800 Kgs Lyngby, Denmark.
authors: Joakim M
authors: JM
authors: Andersen
authors: Enzyme and Protein Chemistry, Department of Systems Biology, Technical University of Denmark, Søltofts Plads, Bldg. 224, DK-2800 Kgs Lyngby, Denmark.
authors: Julie B
authors: JB
authors: Rannes
authors: Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark. Electronic address: abl@plen.ku.dk.
authors: Andreas
authors: A
authors: Blennow
authors: Enzyme and Protein Chemistry, Department of Systems Biology, Technical University of Denmark, Søltofts Plads, Bldg. 224, DK-2800 Kgs Lyngby, Denmark. Electronic address: bis@bio.dtu.dk.
authors: Birte
authors: B
authors: Svensson
mesh_terms: D000327:Adsorption; D000681:Amylases / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D055162:Biocatalysis; D004587:Electrophoresis, Agar Gel; D001467:Hordeum / Q000201:enzymology*; D018613:Microscopy, Confocal; D008958:Models, Molecular; D009154:Mutation; D009844:Oligosaccharides / Q000737:chemistry; D011485:Protein Binding; D013213:Starch / Q000737:chemistry*; D013329:Structure-Activity Relationship; D020349:Surface Plasmon Resonance
chemical_list: D009844:Oligosaccharides; D013213:Starch; D000681:Amylases
keywords: Barley α-amylase; Starch degradation; Starch surface binding