Journal article
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 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.
Language: | English |
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Year: | 2010 |
Pages: | 739-750 |
ISSN: | 10898638 and 00222836 |
Types: | Journal article |
DOI: | 10.1016/j.jmb.2010.09.031 |
ORCIDs: | Abou Hachem, Maher and Svensson, Birte |
Catalytic Domain Crystallography, X-Ray Cyclodextrins Dimerization Glycoside Hydrolases Hordeum Macromolecular Substances Models, Molecular Protein Structure, Quaternary Protein Structure, Tertiary Recombinant Proteins Seeds Starch inhibitor complex structure plant pullulanase pullulanase specificity substrate α-1,6-glucoside debranching