Crystal structures of the starch-binding domain from Rhizopus oryzae glucoamylase reveal a polysaccharide-binding path

2008 ◽  
Vol 416 (1) ◽  
pp. 27-36 ◽  
Author(s):  
Jung-Yu Tung ◽  
Margaret Dah-Tsyr Chang ◽  
Wei-I Chou ◽  
Yen-Yi Liu ◽  
Yi-Hung Yeh ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Hydrophobic Interaction ◽  
Binding Sites ◽  
Catalytic Domain ◽  
Rhizopus Oryzae ◽  
Binding Domain ◽  
Scatchard Analysis ◽  
Carbohydrate Binding ◽  
Binding Isotherm ◽  
Long Chain

GA (glucoamylase) hydrolyses starch and polysaccharides to β-D-glucose. RoGA (Rhizopus oryzae GA) consists of two functional domains, an N-terminal SBD (starch-binding domain) and a C-terminal catalytic domain, which are connected by an O-glycosylated linker. In the present study, the crystal structures of the SBD from RoGA (RoGACBM21) and the complexes with β-cyclodextrin (SBD–βCD) and maltoheptaose (SBD–G7) were determined. Two carbohydrate binding sites, I (Trp47) and II (Tyr32), were resolved and their binding was co-operative. Besides the hydrophobic interaction, two unique polyN loops comprising consecutive asparagine residues also participate in the sugar binding. A conformational change in Tyr32 was observed between unliganded and liganded SBDs. To elucidate the mechanism of polysaccharide binding, a number of mutants were constructed and characterized by a quantitative binding isotherm and Scatchard analysis. A possible binding path for long-chain polysaccharides in RoGACBM21 was proposed.

scholarly journals The family 21 carbohydrate-binding module of glucoamylase from Rhizopus oryzae consists of two sites playing distinct roles in ligand binding

2006 ◽  
Vol 396 (3) ◽  
pp. 469-477 ◽  
Author(s):  
Wei-I Chou ◽  
Tun-Wen Pai ◽  
Shi-Hwei Liu ◽  
Bor-Kai Hsiung ◽  
Margaret D.-T. Chang
Keyword(s):  
Ligand Binding ◽  
Binding Sites ◽  
Catalytic Domain ◽  
Rhizopus Oryzae ◽  
Structural Information ◽  
Molecular Model ◽  
Site Directed Mutagenesis ◽  
Carbohydrate Binding ◽  
Carbohydrate Binding Module ◽  
Ligand Binding Sites

The starch-hydrolysing enzyme GA (glucoamylase) from Rhizopus oryzae is a commonly used glycoside hydrolase in industry. It consists of a C-terminal catalytic domain and an N-terminal starch-binding domain, which belong to the CBM21 (carbohydrate-binding module, family 21). In the present study, a molecular model of CBM21 from R. oryzae GA (RoGACBM21) was constructed according to PSSC (progressive secondary structure correlation), modified structure-based sequence alignment, and site-directed mutagenesis was used to identify and characterize potential ligand-binding sites. Our model suggests that RoGACBM21 contains two ligand-binding sites, with Tyr32 and Tyr67 grouped into site I, and Trp47, Tyr83 and Tyr93 grouped into site II. The involvement of these aromatic residues has been validated using chemical modification, UV difference spectroscopy studies, and both qualitative and quantitative binding assays on a series of RoGACBM21 mutants. Our results further reveal that binding sites I and II play distinct roles in ligand binding, the former not only is involved in binding insoluble starch, but also facilitates the binding of RoGACBM21 to long-chain soluble polysaccharides, whereas the latter serves as the major binding site mediating the binding of both soluble polysaccharide and insoluble ligands. In the present study we have for the first time demonstrated that the key ligand-binding residues of RoGACBM21 can be identified and characterized by a combination of novel bioinformatics methodologies in the absence of resolved three-dimensional structural information.

scholarly journals Crystal structure of GH43 exo-β-1,3-galactanase from the basidiomycete Phanerochaete chrysosporium provides insights into the mechanism of bypassing side chains

2020 ◽  
Author(s):  
Kaori Matsuyama ◽  
Naomi Kishine ◽  
Zui Fujimoto ◽  
Naoki Sunagawa ◽  
Toshihisa Kotake ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Phanerochaete Chrysosporium ◽  
Catalytic Domain ◽  
Interaction Mechanism ◽  
Binding Domain ◽  
Glycoside Hydrolases ◽  
Glycoside Hydrolase Family ◽  
Carbohydrate Binding ◽  
Side Chains ◽  
Ligand Interaction

AbstractArabinogalactan proteins (AGPs) are functional plant proteoglycans, but their functions are largely unexplored, mainly because of the complexity of the sugar moieties, which are generally analyzed with the aid of glycoside hydrolases. In this study, we solved the apo and liganded structures of exo-β-1,3-galactanase from the basidiomycete Phanerochaete chrysosporium (Pc1,3Gal43A), which specifically cleaves AGPs. It is composed of a glycoside hydrolase family 43 subfamily 24 (GH43_sub24) catalytic domain together with a carbohydrate-binding module family (CBM) 35 binding domain. GH43_sub24 lacks the catalytic base Asp that is conserved among other GH43 subfamilies. Crystal structure and kinetic analyses indicated that the tautomerized imidic acid function of Gln263 serves instead as the catalytic base residue. Pc1,3Gal43A has three subsites that continue from the bottom of the catalytic pocket to the solvent. Subsite -1 contains a space that can accommodate the C-6 methylol of Gal, enabling the enzyme to bypass the β-1,6-linked galactan side chains of AGPs. Furthermore, the galactan-binding domain in CBM35 has a different ligand interaction mechanism from other sugar-binding CBM35s. Some of the residues involved in ligand recognition differ from those of galactomannan-binding CBM35, including substitution of Trp for Gly, which affects pyranose stacking, and substitution of Asn for Asp in the lower part of the binding pocket. Pc1,3Gal43A WT and its mutants at residues involved in substrate recognition are expected to be useful tools for structural analysis of AGPs. Our findings should also be helpful in engineering designer enzymes for efficient utilization of various types of biomass.

Crystal structures of the GH6 Orpinomyces sp. Y102 CelC7 enzyme with exo and endo activity and its complex with cellobiose

2019 ◽  
Vol 75 (12) ◽  
pp. 1138-1147
Author(s):  
Hsiao-Chuan Huang ◽  
Liu-Hong Qi ◽  
Yo-Chia Chen ◽  
Li-Chu Tsai
Keyword(s):  
Enzymatic Hydrolysis ◽  
Crystal Structures ◽  
Active Site ◽  
Binding Sites ◽  
Glycoside Hydrolase ◽  
Catalytic Domain ◽  
Glycoside Hydrolase Family ◽  
Substrate Binding Sites ◽  
Key Residues ◽  
Hydrolase Family

The catalytic domain (residues 128–449) of the Orpinomyces sp. Y102 CelC7 enzyme (Orp CelC7) exhibits cellobiohydrolase and cellotriohydrolase activities. Crystal structures of Orp CelC7 and its cellobiose-bound complex have been solved at resolutions of 1.80 and 2.78 Å, respectively. Cellobiose occupies subsites +1 and +2 within the active site of Orp CelC7 and forms hydrogen bonds to two key residues: Asp248 and Asp409. Furthermore, its substrate-binding sites have both tunnel-like and open-cleft conformations, suggesting that the glycoside hydrolase family 6 (GH6) Orp CelC7 enzyme may perform enzymatic hydrolysis in the same way as endoglucanases and cellobiohydrolases. LC-MS/MS analysis revealed cellobiose (major) and cellotriose (minor) to be the respective products of endo and exo activity of the GH6 Orp CelC7.

Crystal structures ofEscherichia colibranching enzyme in complex with cyclodextrins

2016 ◽  
Vol 72 (5) ◽  
pp. 641-647 ◽  
Author(s):  
Lei Feng ◽  
Remie Fawaz ◽  
Stacy Hovde ◽  
Fang Sheng ◽  
Meisam Nosrati ◽  
...  
Keyword(s):  
Binding Site ◽  
Crystal Structures ◽  
Binding Sites ◽  
Carbohydrate Binding ◽  
Binding Modes ◽  
Branch Points ◽  
Branching Enzymes ◽  
Similarities And Differences ◽  
Amp Activated Protein Kinase ◽  
Shed Light

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 ofEscherichia coliBE (EcBE) in complex with α-, β- and γ-cyclodextrin were determined in order to better understand substrate binding. Four cyclodextrin-binding sites were identified inEcBE; 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 ofEcBE 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.

scholarly journals The structure of a glycoside hydrolase 29 family member from a rumen bacterium reveals unique, dual carbohydrate-binding domains

2016 ◽  
Vol 72 (10) ◽  
pp. 750-761 ◽  
Author(s):  
Emma L. Summers ◽  
Christina D. Moon ◽  
Renee Atua ◽  
Vickery L. Arcus
Keyword(s):  
Glycoside Hydrolase ◽  
Catalytic Domain ◽  
Binding Domain ◽  
Carbohydrate Binding ◽  
Catalytic Core ◽  
Binding Domains ◽  
Carbohydrate Binding Modules ◽  
Tim Barrel ◽  
Domain Arrangement ◽  
Hydrolysis Of

Glycoside hydrolase (GH) family 29 consists solely of α-L-fucosidases. These enzymes catalyse the hydrolysis of glycosidic bonds. Here, the structure of GH29_0940, a protein cloned from metagenomic DNA from the rumen of a cow, has been solved, which reveals a multi-domain arrangement that has only recently been identified in bacterial GH29 enzymes. The microbial species that provided the source of this enzyme is unknown. This enzyme contains a second carbohydrate-binding domain at its C-terminal end in addition to the typical N-terminal catalytic domain and carbohydrate-binding domain arrangement of GH29-family proteins. GH29_0940 is a monomer and its overall structure consists of an N-terminal TIM-barrel-like domain, a central β-sandwich domain and a C-terminal β-sandwich domain. The TIM-barrel-like catalytic domain exhibits a (β/α)8/7arrangement in the core instead of the typical (β/α)8topology, with the `missing' α-helix replaced by a long meandering loop that `closes' the barrel structure and suggests a high degree of structural flexibility in the catalytic core. This feature was also noted in all six other structures of GH29 enzymes that have been deposited in the PDB. Based on sequence and structural similarity, the residues Asp162 and Glu220 are proposed to serve as the catalytic nucleophile and the proton donor, respectively. Like other GH29 enzymes, the GH29_0940 structure shows five strictly conserved residues in the catalytic pocket. The structure shows two glycerol molecules in the active site, which have also been observed in other GH29 structures, suggesting that the enzyme catalyses the hydrolysis of small carbohydrates. The two binding domains are classed as family 32 carbohydrate-binding modules (CBM32). These domains have residues involved in ligand binding in the loop regions at the edge of the β-sandwich. The predicted substrate-binding residues differ between the modules, suggesting that different modules bind to different groups on the substrate(s). Enzymes that possess multiple copies of CBMs are thought to have a complex mechanism of ligand recognition. Defined electron density identifying a long 20-amino-acid hydrophilic loop separating the two CBMs was observed. This suggests that the additional C-terminal domain may have a dynamic range of movement enabled by the loop, allowing a unique mode of action for a GH29 enzyme that has not been identified previously.

scholarly journals The Fibronectin Type 3-Like Repeat from the Clostridium thermocellum Cellobiohydrolase CbhA Promotes Hydrolysis of Cellulose by Modifying Its Surface

2002 ◽  
Vol 68 (9) ◽  
pp. 4292-4300 ◽  
Author(s):  
Irina A. Kataeva ◽  
Ronald D. Seidel ◽  
Ashit Shah ◽  
Larry T. West ◽  
Xin-Liang Li ◽  
...  
Keyword(s):  
Filter Paper ◽  
Clostridium Thermocellum ◽  
Catalytic Domain ◽  
Glycosyl Hydrolase ◽  
Binding Domain ◽  
Carbohydrate Binding ◽  
Hydrolysis Of Cellulose ◽  
Type 3 ◽  
Hydrolysis Of ◽  
Fibronectin Type

ABSTRACT Fibronectin type 3 homology domains (Fn3) as found in the cellobiohydrolase CbhA of Clostridium thermocellum are common among bacterial extracellular glycohydrolases. The function of these domains is not clear. CbhA is modular and composed of an N-terminal family IV carbohydrate-binding domain (CBDIV), an immunoglobulin-like domain, a family 9 glycosyl hydrolase catalytic domain (Gh9), two Fn3-like domains (Fn31,2), a family III carbohydrate-binding domain (CBDIII), and a dockerin domain. Efficiency of cellulose hydrolysis by truncated forms of CbhA increased in the following order: Gh9 (lowest efficiency), Gh9-Fn31,2 (more efficient), and Gh9-Fn31,2-CBDIII (greatest efficiency). Thermostability of the above constructs decreased in the following order: Gh9 (most stable), Gh9-Fn31,2, and then Gh9-Fn31,2-CBDIII (least stable). Mixing of Orpinomyces endoglucanase CelE with Fn31,2, or Fn31,2-CBDIII increased efficiency of hydrolysis of acid-swollen cellulose (ASC) and filter paper. Scanning electron microscopic studies of filter paper treated with Fn31,2, Fn31,2-CBDIII, or CBDIII showed that the surface of the cellulose fibers had been loosened up and crenellated by Fn31,2 and Fn31,2-CBDIII and to a lesser extent by CBDIII. X-ray diffraction analysis did not reveal changes in the crystallinity of the filter paper. CBDIII bound to ASC and filter paper with capacities of 2.45 and 0.73 μmoles g−1 and relative affinities (K r) of 1.12 and 2.13 liters g−1, respectively. Fn31,2 bound weakly to both celluloses. Fn31,2-CBD bound to ASC and filter paper with capacities of 3.22 and 0.81 μmoles g−1 and K rs of 1.14 and 1.98 liters g−1, respectively. Fn31,2 and CBDIII contained 2 and 1 mol of calcium per mol, respectively. The results suggest that Fn31,2 aids the hydrolysis of cellulose by modifying its surface. This effect is enhanced by the presence of CBDIII, which increases the concentration of Fn31,2 on the cellulose surface.

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