Crystal structures ofEscherichia colibranching enzyme in complex with cyclodextrins

Abstract

Branching enzyme (BE) is responsible for the third step in glycogen/starch biosynthesis. It catalyzes the cleavage of α-1,4 glucan linkages and subsequent reattachment to form α-1,6 branch points. These branches are crucial to the final structure of glycogen and starch. The crystal structures of Escherichia coli BE (EcBE) in complex with α-, β- and γ-cyclodextrin were determined in order to better understand substrate binding. Four cyclodextrin-binding sites were identified in EcBE; they were all located on the surface of the enzyme, with none in the vicinity of the active site. While three of the sites were also identified as linear polysaccharide-binding sites, one of the sites is specific for cyclodextrins. In previous work three additional binding sites were identified as exclusively binding linear malto-oligosaccharides. Comparison of the binding sites shed light on this apparent specificity. Binding site IV is located in the carbohydrate-binding module 48 (CBM48) domain of EcBE and superimposes with the cyclodextrin-binding site found in the CBM48 domain of 5'-AMP-activated protein kinase (AMPK). Comparison of these sites shows the similarities and differences in the two binding modes. While some of the binding sites were found to be conserved between branching enzymes of different organisms, some are quite divergent, indicating both similarities and differences between oligosaccharide binding in branching enzymes from various sources.

Identification and characterization of a novel starch branching enzyme from the picoalgae Ostreococcus tauri

“…The comparison between the OsttaBE model of O. sativa and E. coli BEs showed that the global topology is almost the same ( Figure 3); however, a closer view of the superposed catalytic sites exhibit a high spatial conservation of the amino acids involved in catalysis (Asp647, Glu684, and Asp816, OsttaBE numbering) with those from the bacterial enzyme ( Figure 3B). In addition, we also observed a conservation of other three essential residues involved in catalysis previously identified in the E. coli BE as Tyr300, Asp335 and Asp526 [57,73] at 531, 573 and 816 positions in OsttaBE, respectively ( Figure 3). The importance of Tyr300 for the stability and catalysis and in E. coli BE had already been described by MacGregor and Svensson [74] and Matsui et al [75].…”

“…OsttaBE structure was modeled using starch debranching enzyme from Hordeum vulgare (PDB code 4J3S)[54]. Oryza sativa and E. coli BE crystal structure´s (PDB codes 3AMK and 5E6Z, respectively) were used to compare catalytic domains[55][56][57]. Alignment with the template was based on homology and secondary structure.…”

Bound Substrate in the Structure of Cyanobacterial Branching Enzyme Supports a New Mechanistic Model

“…Binding of oligosaccharides in the active site cleft of BE was observed for the first time in the present study. In previous studies, sugar chains were found only at certain distances from the active site in the crystal structures (OsBEI (23), HsBE (22), and EcBE (24,25)). Binding of the glucan chain at the active site was accomplished by soaking the protein crystals of cceBE1 in high concentrations of oligosaccharides, namely 500 mM G6 and 300 mM G7.…”

“…These studies provided basic information on the overall and domain structures of GH13_9 (prokaryotic)-and GH13_8 (eukaryotic)-type BEs. The structures of OsBEI (23), HsBE (22), and EcBE (24,25) in complex with linear maltooligosaccharides or cyclodextrins led to the identification of surface binding sites (SBSs) located on the enzyme surface at certain distances from the active site (26 -29). Possible roles of SBSs (substrate targeting, guiding substrate into the active site, and passing on reactions products) have been proposed (27).…”

Cyclodextrin Enhances Corneal Tolerability and Reduces Ocular Toxicity Caused by Diclofenac

“…In order to predict the orientation within the cavity and/or rim and gain more insights into the stability/binding constants of Diclo with α -CD, β -CD, γ -CD, and HP- β -CD, molecular docking studies were performed using Molecular Operating Environment (MOE) 2014.09 software (Chemical Computing Group, Montreal, QC, Canada). The crystal structures of CDs were extracted from Protein Data Bank (PDB): α -CD (PDB code: 5E6Y), β -CD (PDB code: 5E6Z), and γ -CD (PDB code: 5E70) [ 19 ]. Since no crystal structure is available for HP- β -CD, the crystal structure of β -CD (PDB code: 5E6Z) was used as a template to build the 3D structure of HP- β -CD by substituting four primary hydroxyl groups with 2-hydroxypropyl radical, as described elsewhere [ 20 ].…”