The Structure of Barley α-Amylase Isozyme 1 Reveals a Novel Role of Domain C in Substrate Recognition and Binding

Robert, Xavier; Haser, R.; Gottschalk, Tine E.; Ratajczak, Fabien; Driguez, Hugues; Svensson, Birte; Aghajari, Nushin

doi:10.1016/s0969-2126(03)00151-5

Cited by 137 publications

(172 citation statements)

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“…Several α-amylases are described to possess this type of binding sites (Gibson & Svensson 1987;Larson et al 1994;Kadziola et al 1998;Dauter et al 1999;Brzozowski et al 2000;Ramasubbu et al 2003;Robert et al 2003Robert et al , 2005Lyhne-Iversen et al 2006;Vujicic-Žagar & Dijkstra, 2006;Ragunath et al 2008). In barley α-amylase isozyme 1 (AMY1), the crystal structure of the inactive catalytic nucleophile D180A mutant in complex with maltoheptaose ( Fig.…”

Section: Impact Of Secondary Binding Sites On Functionmentioning

confidence: 99%

“…6) highlighted oligosaccharide binding at two external surface sites and the active site, respectively (Robert et al 2005). One surface site, called "the pair of sugar tongs", was situated on the non-catalytic C-terminal domain, while the other was found on the side of the catalytic (β/α) 8 -barrel at a certain distance from the active site (Robert et al 2003(Robert et al , 2005. The chain direction of the bound oligosaccharides in the D180A AMY1/maltoheptaose complex was such that the three molecules could not be visualized to all belong to the same polysaccharide molecule (Robert et al 2005).…”

Section: Impact Of Secondary Binding Sites On Functionmentioning

confidence: 99%

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An enzyme family reunion — similarities, differences and eccentricities in actions on α-glucans

et al. 2008

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α-Glucans in general, including starch, glycogen and their derived oligosaccharides are processed by a host of more or less closely related enzymes that represent wide diversity in structure, mechanism, specificity and biological role. Sophisticated three-dimensional structures continue to emerge hand-in-hand with the gaining of novel insight in modes of action. We are witnessing the "test of time" blending with remaining questions and new relationships for these enzymes. Information from both within and outside of ALAMY 3 Symposium will provide examples on what the family contains and outline some future directions. In 2007 a quantum leap crowned the structural biology by the glucansucrase crystal structure. This initiates the disclosure of the mystery on the organisation of the multidomain structure and the "robotics mechanism" of this group of enzymes. The central issue on architecture and domain interplay in multidomain enzymes is also relevant in connection with the recent focus on carbohydrate-binding domains as well as on surface binding sites and their long underrated potential. Other questions include, how different or similar are glycoside hydrolase families 13 and 31 and is the lid finally lifted off the disguise of the starch lyase, also belonging to family 31? Is family 57 holding back secret specificities? Will the different families be sporting new "eccentric" functions, are there new families out there, and why are crystal structures of "simple" enzymes still missing? Indeed new understanding and discovery of biological roles continuously emphasize value of the collections of enzyme models, sequences, and evolutionary trees which will also be enabling advancement in design for useful and novel applications.

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Section: Impact Of Secondary Binding Sites On Functionmentioning

confidence: 99%

Section: Impact Of Secondary Binding Sites On Functionmentioning

confidence: 99%

An enzyme family reunion — similarities, differences and eccentricities in actions on α-glucans

et al. 2008

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“…Indeed, the structure and biochemistry of several family 20 CBMs, which bind to starch, have been analysed extensively (see [6][7][8][9][10][11][12][13] for examples). Furthermore, numerous crystal structures of starch-modifying enzymes have revealed malto-oligosaccharide-binding sites that are distinct from the substrate-binding cleft, indicating that these enzymes also contain starch-binding CBMs [14][15][16][17].…”

Section: Introductionmentioning

confidence: 99%

Carbohydrate-binding modules: fine-tuning polysaccharide recognition

Boraston

Bolam

Gilbert

et al. 2004

Biochemical Journal

1,777

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The enzymic degradation of insoluble polysaccharides is one of the most important reactions on earth. Despite this, glycoside hydrolases attack such polysaccharides relatively inefficiently as their target glycosidic bonds are often inaccessible to the active site of the appropriate enzymes. In order to overcome these problems, many of the glycoside hydrolases that utilize insoluble substrates are modular, comprising catalytic modules appended to one or more non-catalytic CBMs (carbohydrate-binding modules). CBMs promote the association of the enzyme with the substrate. In view of the central role that CBMs play in the enzymic hydrolysis of plant structural and storage polysaccharides, the ligand specificity displayed by these protein modules and the mechanism by which they recognize their target carbohydrates have received considerable attention since their discovery almost 20 years ago. In the last few years, CBM research has harnessed structural, functional and bioinformatic approaches to elucidate the molecular determinants that drive CBM-carbohydrate recognition. The present review summarizes the impact structural biology has had on our understanding of the mechanisms by which CBMs bind to their target ligands.

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“…In the barley α amylases three structural calcium ions are present. 20,21) They superimpose perfectly in AMY1 and AMY2 and also share all protein ligands. Calcium, however, has distinctly different effects on stability and enzymatic activity of AMY1 and AMY2 and the isozymes, therefore, represent a unique source to understand structural features that modulate the impact of calcium on the behaviour of α amylases.…”

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confidence: 84%

“…3). 20,21) Ca500 is at a distance of 7 A from another Ca 2+ (Ca502) with ligands only in domain B, including the common ligand with Ca500 (Asp149AMY1, Asp148AMY2). Ca501 has all ligands coming from a short loop in domain B (Phe108AMY1 Asp118AMY1) and has also two water molecules as ligands.…”

Section: Role Of Calcium Ions In Amy1 and Amy2 Stability And Activitymentioning

confidence: 99%

Interactions between Barley .ALPHA.-Amylases, Substrates, Inhibitors and Regulatory Proteins

et al. 2006

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α Amylases belong to the glycoside hydrolase family 13 (GH13) that together with glycoside hydrolase families 70 and 77 constitute clan H of glycoside hydrolases (GH H).1) GH H contains approximately 30 different enzyme specificities acting in hydrolase and transglucosidase reactions on α 1,4 and or α 1,6 glucan and α glucoside substrates.2)The three dimensional structures of α amylases mostly contain three domains; domain A, a (β α)8 barrel; domain B a small protruding loop situated between β strand and α helix 3 in the (β α)8 barrel; and domain C, a C terminal anti parallel β sheet composed of 5 10 strands. The substrate binding site is made from residues of domains A and B and the three acid residues involved in catalysis are situated at the C terminal ends of β strands 4, 5 and 7.2) These three catalytic residues are the only invariant residues in GH H.3) A few new complexes between amylolytic enzymes and substrates or substrate analogues have been recently published. 4,5) These structures provide novel insight in particular with respect to Abstract: Barley α-amylase binds sugars at two sites on the enzyme surface in addition to the active site. Crystallography and site-directed mutagenesis highlight the importance of aromatic residues at these surface sites as demonstrated by Kd values determined for β-cyclodextrin by surface plasmon resonance and for starch granules by adsorption analysis. Activity towards amylopectin and amylose follows two different kinetic models, degradation of amylopectin being composed of a fast and a slow component, perhaps reflecting attack on A and B chains, respectively, whereas amylose hydrolysis follows a simple Michaelian kinetics. β-cyclodextrin binding at surface sites inhibits only the fast reaction in amylopectin degradation. Site-directed mutagenesis and activity analysis, furthermore show that one of the surface binding sites as well as individual subsites in the active site cleft have distinct roles in the multiple attack on amylose. Although the two isozymes AMY1 and AMY2 share ligands for three structural calcium ions, they differ importantly in the effect of calcium on activity and stability, AMY1 having the higher affinity and the lower stability. The role of the individual calcium ions is studied by mutagenesis, crystallography and microcalorimetry. Further improvement of recombinant AMY2 production allows future direct mutational analysis in this isozyme. Specific proteinaceous inhibitors act on α-amylases of different origin. In the complex of barley α-amylase subtilisin inhibitor (BASI) with AMY2, a fully hydrated calcium ion at the protein interface mediates contact between inhibitor residues and the enzyme catalytic groups in a manner that depends on calcium and which can be suppressed by site-directed mutagenesis of Glu168 in BASI. Finally certain inhibitors and enzymes are targets of the disulphide reductase thioredoxin h that attacks a specific disulphide bond in BASI and, remarkably, reduces two different disulphide bonds in the barley monomeric and dimeri...

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The Structure of Barley α-Amylase Isozyme 1 Reveals a Novel Role of Domain C in Substrate Recognition and Binding

Cited by 137 publications

References 60 publications

An enzyme family reunion — similarities, differences and eccentricities in actions on α-glucans

An enzyme family reunion — similarities, differences and eccentricities in actions on α-glucans

Carbohydrate-binding modules: fine-tuning polysaccharide recognition

Interactions between Barley .ALPHA.-Amylases, Substrates, Inhibitors and Regulatory Proteins

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