PDBsum entry 1mxg

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Hydrolase PDB id
Protein chain
435 a.a. *
ACR ×3
ETE ×2
EOH ×4
_MG ×3
Waters ×509
* Residue conservation analysis

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Key reference
Title Differential regulation of a hyperthermophilic alpha-Amylase with a novel (ca,Zn) two-Metal center by zinc.
Authors A.Linden, O.Mayans, W.Meyer-Klaucke, G.Antranikian, M.Wilmanns.
Ref. J Biol Chem, 2003, 278, 9875-9884. [DOI no: 10.1074/jbc.M211339200]
PubMed id 12482867
The crystal structure of the alpha-amylase from the hyperthermophilic archaeon Pyrococcus woesei was solved in the presence of three inhibitors: acarbose, Tris, and zinc. In the absence of exogenous metals, this alpha-amylase bound 1 and 4 molar eq of zinc and calcium, respectively. The structure reveals a novel, activating, two-metal (Ca,Zn)-binding site and a second inhibitory zinc-binding site that is found in the -1 sugar-binding pocket within the active site. The data resolve the apparent paradox between the zinc requirement for catalytic activity and its strong inhibitory effect when added in molar excess. They provide a rationale as to why this alpha-amylase, in contrast to commercially available alpha-amylases, does not require the addition of metal ions for full catalytic activity, suggesting it as an ideal target to maximize the efficiency of industrial processes like liquefaction of starch.
Figure 1.
Fig. 1. Overall crystal structure of PWA complexed with different ligands. The cylinders and arrows represent helices and -strands, respectively. Domains A-C are colored cyan, magenta, and brown, respectively. For clarity, the -strands composing the -barrel of domain A are colored gray. Bound metals (zinc, green; and magnesium and calcium, orange) and acarbose molecules are indicated and numbered according to Table II. The backbone of the acarbose molecules is yellow; oxygen atoms are red; and nitrogen atoms are blue. A, top view of the PWA·Ac/Zn complex. The active site cleft at the front face of the PWA molecule contains two inhibitors with partial occupancies, the first of which is acarbose and the second of which is a coordinated zinc ion, which is virtually identical to the nitrogen position of the 4-amino-4,6-dideoxy- -D-glucose ring of acarbose. The two inhibitors are superimposed onto each other. B, PWA·Ac/Zn rotated 90° along a horizontal axis with respect to the orientation in A. C, PWA·Tris shown from the same orientation as in A. Three zinc-binding sites (Zn3, Zn5, and Zn6) of PWA·Ac/Zn are replaced by magnesium ions in PWA·Tris (Mg3, Mg5, and Mg6). In PWA·Tris, no metal is found in site 4 of the PWA·Ac/Zn structure.
Figure 3.
Fig. 3. Active site of PWA with bound acarbose and zinc (PWA·Ac/Zn; A and B), Tris (PWA·Tris; C and D), and zinc (PWA·Zn; E and F). A, C, and E, structures of active site residues in the presence of bound ligands. Each F[o] F[c] difference electron density map (green) in the absence of ligands (A, acarbose; C, Tris; and E, zinc) is contoured at 2.0, 2.0, and 4.5 , respectively. In E, the anomalous difference peak (red) is contoured at 3.7 . B, D, and F, schematic representations of the ligands bound to the active site. Hydrogen bonds are shown by dashed lines. Zinc ions and solvent molecules are shown as green and gray spheres, respectively. Solvent molecules mediating protein-inhibitor interactions are indicated in B, D, and F; for clarity, Tyr62, Phe^159, and Tyr199 are not shown.
The above figures are reprinted by permission from the ASBMB: J Biol Chem (2003, 278, 9875-9884) copyright 2003.
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