title: Bound Substrate in the Structure of Cyanobacterial Branching Enzyme Supports a New Mechanistic Model.
abstract: Branching enzyme (BE) catalyzes the formation of α-1,6-glucosidic linkages in amylopectin and glycogen. The reaction products are variable, depending on the organism sources, and the mechanistic basis for these different outcomes is unclear. Although most cyanobacteria have only one BE isoform belonging to glycoside hydrolase family 13,Cyanothecesp. ATCC 51142 has three isoforms (BE1, BE2, and BE3) with distinct enzymatic properties, suggesting that investigations of these enzymes might provide unique insights into this system. Here, we report the crystal structure of ligand-free wild-type BE1 (residues 5-759 of 1-773) at 1.85 Å resolution. The enzyme consists of four domains, including domain N, carbohydrate-binding module family 48 (CBM48), domain A containing the catalytic site, and domain C. The central domain A displays a (β/α)8-barrel fold, whereas the other domains adopt β-sandwich folds. Domain N was found in a new location at the back of the protein, forming hydrogen bonds and hydrophobic interactions with CBM48 and domain A. Site-directed mutational analysis identified a mutant (W610N) that bound maltoheptaose with sufficient affinity to enable structure determination at 2.30 Å resolution. In this structure, maltoheptaose was bound in the active site cleft, allowing us to assign subsites -7 to -1. Moreover, seven oligosaccharide-binding sites were identified on the protein surface, and we postulated that two of these in domain A served as the entrance and exit of the donor/acceptor glucan chains, respectively. Based on these structures, we propose a substrate binding model explaining the mechanism of glycosylation/deglycosylation reactions catalyzed by BE.
authors.affiliation: From the Department of Biological Production, Faculty of Bioresource Sciences, Akita Prefectural University, Shimoshinjyo-Nakano, Akita 010-0195, Japan and.
authors.forename: Mari
authors.initials: M
authors.lastname: Hayashi
authors.affiliation: From the Department of Biological Production, Faculty of Bioresource Sciences, Akita Prefectural University, Shimoshinjyo-Nakano, Akita 010-0195, Japan and ryuichi@akita-pu.ac.jp.
authors.forename: Ryuichiro
authors.initials: R
authors.lastname: Suzuki
authors.affiliation: the Unité de Glycobiologie Structurale et Fonctionnelle, Unité Mixte de Recherche 8576, CNRS-Université des Sciences et Technologies de Lille, 59655 Villeneuve d'Ascq Cedex, France.
authors.forename: Christophe
authors.initials: C
authors.lastname: Colleoni
authors.affiliation: the Unité de Glycobiologie Structurale et Fonctionnelle, Unité Mixte de Recherche 8576, CNRS-Université des Sciences et Technologies de Lille, 59655 Villeneuve d'Ascq Cedex, France.
authors.forename: Steven G
authors.initials: SG
authors.lastname: Ball
authors.affiliation: From the Department of Biological Production, Faculty of Bioresource Sciences, Akita Prefectural University, Shimoshinjyo-Nakano, Akita 010-0195, Japan and.
authors.forename: Naoko
authors.initials: N
authors.lastname: Fujita
authors.affiliation: From the Department of Biological Production, Faculty of Bioresource Sciences, Akita Prefectural University, Shimoshinjyo-Nakano, Akita 010-0195, Japan and esuzuki@akita-pu.ac.jp.
authors.forename: Eiji
authors.initials: E
authors.lastname: Suzuki
mesh_terms: D015061:1,4-alpha-Glucan Branching Enzyme / Q000737:chemistry* / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry; D020134:Catalytic Domain; D003460:Crystallization; D000458:Cyanobacteria; D046889:Cyanothece / Q000737:chemistry*; D005936:Glucans / Q000378:metabolism; D006031:Glycosylation; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011487:Protein Conformation; D000072417:Protein Domains; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D005936:Glucans; C031146:maltoheptaose; D015061:1,4-alpha-Glucan Branching Enzyme
keywords: branching enzyme; carbohydrate biosynthesis; cyanobacteria; enzyme mechanism; enzyme structure; glycogen; starch; surface binding site