Crystal Structure of an Essential Enzyme in Seed Starch Degradation: Barley Limit Dextrinase in Complex with Cyclodextrins |
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Authors: | Malene Bech Vester-Christensen Maher Abou Hachem Anette Henriksen |
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Institution: | 1 Enzyme and Protein Chemistry, Department of Systems Biology, Søltofts Plads, Building 224, Technical University of Denmark, DK-2800 Kgs. Lyngby, Denmark2 Protein Chemistry Group, Carlsberg Laboratory, Carlsberg Research Center, Gamle Carlsberg Vej 10, DK-2500 Valby, Denmark |
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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. |
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Keywords: | HvLD Hordeum vulgare limit dextrinase CD cyclodextrin Glc glucose LD limit dextrinase GH glycoside hydrolase KpPUL Klebsiella pneumoniae pullulanase CBM carbohydrate binding module BaPUL Bacillus acidopullulyticuspullulanase Gol glycerol GBD glycogen binding domain G4 maltotetraose PDB Protein Data Bank MR molecular replacement BsPUL Bacillus subtilis str 168 pullulanase PaISO Pseudomonas amyloderamosa isoamylase PEG polyethylene glycol ESRF European Synchrotron Radiation Facility |
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