Crystal Structure of an Essential Enzyme in Seed Starch Degradation: Barley Limit Dextrinase in Complex with Cyclodextrins

Barley limit dextrinase [Hordeum vulgare limit dextrinase (HvLD)] catalyzes the hydrolysis of alpha-1,6 glucosidic linkages m limit dextrins This activity plays a role m starch degradation during germination and presumably in starch biosynthesis during gram filling The crystal structures of HvLD m complex with the competitive inhibitors alpha-cyclodextrin (CD) and beta-CD are solved and refined to 2 5 angstrom and 2 1 angstrom, 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 beta-sandwich domain, a six-stranded beta-sandwich domain belonging to the carbohydrate binding module 48 family, a catalytic (beta/alpha)(8)-like barrel domain that lacks alpha-helix 5, and a C-terminal eight-stranded beta-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 alpha-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 beta 5 and beta 6 of the catalytic domain also seems to influence substrate specificity and to give HvLD a higher affinity for alpha-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 (C) 2010 Elsevier Ltd All rights reserved