title: Crystal structure of the Chlamydomonas starch debranching enzyme isoamylase ISA1 reveals insights into the mechanism of branch trimming and complex assembly.
abstract: The starch debranching enzymes isoamylase 1 and 2 (ISA1 and ISA2) are known to exist in a large complex and are involved in the biosynthesis and crystallization of starch. It is suggested that the function of the complex is to remove misplaced branches of growing amylopectin molecules, which would otherwise prevent the association and crystallization of adjacent linear chains. Here, we investigate the function of ISA1 and ISA2 from starch producing alga Chlamydomonas. Through complementation studies, we confirm that the STA8 locus encodes for ISA2 and sta8 mutants lack the ISA1·ISA2 heteromeric complex. However, mutants retain a functional dimeric ISA1 that is able to partly sustain starch synthesis in vivo. To better characterize ISA1, we have overexpressed and purified ISA1 from Chlamydomonas reinhardtii (CrISA1) and solved the crystal structure to 2.3 Å and in complex with maltoheptaose to 2.4 Å. Analysis of the homodimeric CrISA1 structure reveals a unique elongated structure with monomers connected end-to-end. The crystal complex reveals details about the mechanism of branch binding that explains the low activity of CrISA1 toward tightly spaced branches and reveals the presence of additional secondary surface carbohydrate binding sites.
authors.affiliation: Carlsberg Laboratory, Gamle Carlsberg Vej 10, DK-1799 Copenhagen V, Denmark and. 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 and.
authors.forename: Sophie R
authors.initials: SR
authors.lastname: Beeren
authors.affiliation: Unité de Glycobiologie Structurale et Fonctionnelle, UMR 8576 CNRS-USTL, Bâtiment C9, Cité Scientifique, F-59655 Villeneuve d'Ascq, France.
authors.forename: Justin
authors.initials: J
authors.lastname: Findinier
authors.affiliation: Unité de Glycobiologie Structurale et Fonctionnelle, UMR 8576 CNRS-USTL, Bâtiment C9, Cité Scientifique, F-59655 Villeneuve d'Ascq, France.
authors.forename: David
authors.initials: D
authors.lastname: Dauvillée
authors.affiliation: Unité de Glycobiologie Structurale et Fonctionnelle, UMR 8576 CNRS-USTL, Bâtiment C9, Cité Scientifique, F-59655 Villeneuve d'Ascq, France.
authors.forename: Steven G
authors.initials: SG
authors.lastname: Ball
authors.affiliation: Carlsberg Laboratory, Gamle Carlsberg Vej 10, DK-1799 Copenhagen V, Denmark and.
authors.forename: Anette
authors.initials: A
authors.lastname: Henriksen
authors.affiliation: Carlsberg Laboratory, Gamle Carlsberg Vej 10, DK-1799 Copenhagen V, Denmark and.
authors.forename: Monica M
authors.initials: MM
authors.lastname: Palcic
mesh_terms: D016825:Chlamydomonas reinhardtii / Q000201:enzymology*; D018360:Crystallography, X-Ray; D005936:Glucans / Q000737:chemistry*; D007517:Isoamylase / Q000737:chemistry*; D010940:Plant Proteins / Q000737:chemistry*; D017434:Protein Structure, Tertiary
chemical_list: D005936:Glucans; D010940:Plant Proteins; C031146:maltoheptaose; D007517:Isoamylase
keywords: Carbohydrate Biosynthesis; Chlamydomonas; Crystal Structure; Debranching Enzyme; Glycosidase; Isoamylase; Polysaccharide; Starch Metabolism; X-ray Crystallography