title: Crystal structure of an essential enzyme in seed starch degradation: barley limit dextrinase in complex with cyclodextrins.
abstract: Barley limit dextrinase [Hordeum vulgare limit dextrinase (HvLD)] catalyzes the hydrolysis of α-1,6 glucosidic linkages in limit dextrins. This activity plays a role in starch degradation during germination and presumably in starch biosynthesis during grain filling. The crystal structures of HvLD in complex with the competitive inhibitors α-cyclodextrin (CD) and β-CD are solved and refined to 2.5 Å and 2.1 Å, respectively, and are the first structures of a limit dextrinase. HvLD belongs to glycoside hydrolase 13 family and is composed of four domains: an immunoglobulin-like N-terminal eight-stranded β-sandwich domain, a six-stranded β-sandwich domain belonging to the carbohydrate binding module 48 family, a catalytic (β/α)(8)-like barrel domain that lacks α-helix 5, and a C-terminal eight-stranded β-sandwich domain of unknown function. The CDs are bound at the active site occupying carbohydrate binding subsites +1 and +2. A glycerol and three water molecules mimic a glucose residue at subsite -1, thereby identifying residues involved in catalysis. The bulky Met440, a unique residue at its position among α-1,6 acting enzymes, obstructs subsite -4. The steric hindrance observed is proposed to affect substrate specificity and to cause a low activity of HvLD towards amylopectin. An extended loop (Asp513-Asn520) between β5 and β6 of the catalytic domain also seems to influence substrate specificity and to give HvLD a higher affinity for α-CD than pullulanases. The crystal structures additionally provide new insight into cation sites and the concerted action of the battery of hydrolytic enzymes in starch degradation.
authors.affiliation: Enzyme and Protein Chemistry, Department of Systems Biology, Søltofts Plads, Building 224, Technical University of Denmark, Kgs. Lyngby, Denmark.
authors.forename: Malene Bech
authors.initials: MB
authors.lastname: Vester-Christensen
authors.forename: Maher
authors.initials: M
authors.lastname: Abou Hachem
authors.forename: Birte
authors.initials: B
authors.lastname: Svensson
authors.forename: Anette
authors.initials: A
authors.lastname: Henriksen
mesh_terms: D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D003505:Cyclodextrins / Q000737:chemistry / Q000378:metabolism*; D019281:Dimerization; D006026:Glycoside Hydrolases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D001467:Hordeum / Q000201:enzymology*; D046911:Macromolecular Substances; D008958:Models, Molecular; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D012639:Seeds / Q000378:metabolism; D013213:Starch / Q000378:metabolism*
chemical_list: D003505:Cyclodextrins; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D013213:Starch; D006026:Glycoside Hydrolases; C100181:pullulanase