title: Oligosaccharide and substrate binding in the starch debranching enzyme barley limit dextrinase.
abstract: Complete hydrolytic degradation of starch requires hydrolysis of both the α-1,4- and α-1,6-glucosidic bonds in amylopectin. Limit dextrinase (LD) is the only endogenous barley enzyme capable of hydrolyzing the α-1,6-glucosidic bond during seed germination, and impaired LD activity inevitably reduces the maltose and glucose yields from starch degradation. Crystal structures of barley LD and active-site mutants with natural substrates, products and substrate analogues were sought to better understand the facets of LD-substrate interactions that confine high activity of LD to branched maltooligosaccharides. For the first time, an intact α-1,6-glucosidically linked substrate spanning the active site of a LD or pullulanase has been trapped and characterized by crystallography. The crystal structure reveals both the branch and main-chain binding sites and is used to suggest a mechanism for nucleophilicity enhancement in the active site. The substrate, product and analogue complexes were further used to outline substrate binding subsites and substrate binding restraints and to suggest a mechanism for avoidance of dual α-1,6- and α-1,4-hydrolytic activity likely to be a biological necessity during starch synthesis.
authors.affiliation: Carlsberg Laboratory, Gamle Carlsberg Vej 10, DK-1799 Copenhagen V, Denmark; Enzyme and Protein Chemistry, Department of Systems Biology, Technical University of Denmark, Søltofts Plads, Building 224, DK-2800 Kongens Lyngby, Denmark.
authors.forename: Marie S
authors.initials: MS
authors.lastname: Møller
authors.affiliation: Carlsberg Laboratory, Gamle Carlsberg Vej 10, DK-1799 Copenhagen V, Denmark. Electronic address: windahl@ruc.dk.
authors.forename: Michael S
authors.initials: MS
authors.lastname: Windahl
authors.affiliation: Carlsberg Laboratory, Gamle Carlsberg Vej 10, DK-1799 Copenhagen V, Denmark. Electronic address: lya.sim@gmail.com.
authors.forename: Lyann
authors.initials: L
authors.lastname: Sim
authors.affiliation: Carlsberg Laboratory, Gamle Carlsberg Vej 10, DK-1799 Copenhagen V, Denmark. Electronic address: marie_boejstrup@hotmail.com.
authors.forename: Marie
authors.initials: M
authors.lastname: Bøjstrup
authors.affiliation: Enzyme and Protein Chemistry, Department of Systems Biology, Technical University of Denmark, Søltofts Plads, Building 224, DK-2800 Kongens Lyngby, Denmark.
authors.forename: Maher
authors.initials: M
authors.lastname: Abou Hachem
authors.affiliation: Carlsberg Laboratory, Gamle Carlsberg Vej 10, DK-1799 Copenhagen V, Denmark. Electronic address: hindsgaulole@gmail.com.
authors.forename: Ole
authors.initials: O
authors.lastname: Hindsgaul
authors.affiliation: Carlsberg Laboratory, Gamle Carlsberg Vej 10, DK-1799 Copenhagen V, Denmark. Electronic address: monicapalcic@gmail.com.
authors.forename: Monica
authors.initials: M
authors.lastname: Palcic
authors.affiliation: Enzyme and Protein Chemistry, Department of Systems Biology, Technical University of Denmark, Søltofts Plads, Building 224, DK-2800 Kongens Lyngby, Denmark. Electronic address: bis@bio.dtu.dk.
authors.forename: Birte
authors.initials: B
authors.lastname: Svensson
authors.affiliation: Carlsberg Laboratory, Gamle Carlsberg Vej 10, DK-1799 Copenhagen V, Denmark. Electronic address: anette.henriksen@gmail.com.
authors.forename: Anette
authors.initials: A
authors.lastname: Henriksen
mesh_terms: D001665:Binding Sites; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000378:metabolism*; D001467:Hordeum / Q000201:enzymology*; D006868:Hydrolysis; D008320:Maltose / Q000378:metabolism*; D008958:Models, Molecular; D009844:Oligosaccharides / Q000378:metabolism*; D011487:Protein Conformation; D013213:Starch / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D009844:Oligosaccharides; C021705:maltooligosaccharides; D008320:Maltose; D013213:Starch; D006026:Glycoside Hydrolases; C100181:pullulanase
keywords: pullulanase; substrate specificity; thio-oligosaccharide; transglycosylase; α-1,6-glucosidase