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DOI no:
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J Biol Chem
278:9875-9884
(2003)
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PubMed id:
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Differential regulation of a hyperthermophilic alpha-amylase with a novel (Ca,Zn) two-metal center by zinc.
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A.Linden,
O.Mayans,
W.Meyer-Klaucke,
G.Antranikian,
M.Wilmanns.
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ABSTRACT
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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.
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Selected figure(s)
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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.
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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.
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The above figures are
reprinted
by permission from the ASBMB:
J Biol Chem
(2003,
278,
9875-9884)
copyright 2003.
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Figures were
selected
by an automated process.
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434 a.a.
_CA
_ZN
