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PDBsum entry 1sci
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Contents |
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* Residue conservation analysis
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References listed in PDB file
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Key reference
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Title
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Reaction mechanism of hydroxynitrile lyases of the alpha/beta-Hydrolase superfamily: the three-Dimensional structure of the transient enzyme-Substrate complex certifies the crucial role of lys236.
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Authors
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K.Gruber,
G.Gartler,
B.Krammer,
H.Schwab,
C.Kratky.
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Ref.
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J Biol Chem, 2004,
279,
20501-20510.
[DOI no: ]
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PubMed id
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Abstract
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The hydroxynitrile lyases (HNLs) from Hevea brasiliensis (HbHNL) and from
Manihot esculenta (MeHNL) are both members of the alpha/beta-hydrolase
superfamily. Mechanistic proposals have been put forward in the past for both
enzymes; they differed with respect to the role of the active-site lysine
residue for which a catalytic function was claimed for the Hevea enzyme but
denied for the Manihot enzyme. We applied a freeze-quench method to prepare
crystals of the complex of HbHNL with the biological substrate acetone
cyanohydrin and determined its three-dimensional structure. Site-directed
mutagenesis was used to prepare the mutant K236L, which is inactive although its
three-dimensional structure is similar to the wild-type enzyme. However, the
structure of the K236L-acetone cyanohydrin complex shows the substrate in a
different orientation from the wild-type complex. Finite difference
Poisson-Boltzmann calculations show that in the absence of Lys(236) the
catalytic base His(235) would be protonated at neutral pH. All of this suggests
that Lys(236) is instrumental for catalysis in several ways, i.e. by correctly
positioning the substrate, by stabilizing the negatively charged reaction
product CN(-), and by modulating the basicity of the catalytic base. These data
complete the elucidation of the reaction mechanism of alpha/beta-hydrolase HNLs,
in which the catalytic triad acts as a general base rather than as a
nucleophile; proton abstraction from the substrate is performed by the serine,
and reprotonation of the product cyanide is performed by the histidine residues.
Together with a threonine side chain, the active-site serine and lysine are also
involved in substrate binding.
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Figure 2.
FIG. 2. The proposed mechanism of the reaction catalyzed by
HbHNL formulated for the cyanohydrin cleavage direction (21).
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Figure 3.
FIG. 3. The mechanism proposed for the reaction catalyzed
by MeHNL (24).
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The above figures are
reprinted
by permission from the ASBMB:
J Biol Chem
(2004,
279,
20501-20510)
copyright 2004.
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Secondary reference #1
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Title
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Atomic resolution crystal structure of hydroxynitrile lyase from hevea brasiliensis.
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Authors
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K.Gruber,
M.Gugganig,
U.G.Wagner,
C.Kratky.
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Ref.
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Biol Chem, 1999,
380,
993-1000.
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PubMed id
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Secondary reference #2
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Title
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Three-Dimensional structures of enzyme-Substrate complexes of the hydroxynitrile lyase from hevea brasiliensis.
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Authors
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J.Zuegg,
K.Gruber,
M.Gugganig,
U.G.Wagner,
C.Kratky.
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Ref.
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Protein Sci, 1999,
8,
1990-2000.
[DOI no: ]
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PubMed id
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Figure 5.
Fig. 5. Residual density within the active site of Hb--HNL for the ~A! F6-acetone, ~B! rhodanide, and ~C! acetone soaks. See caption
to Figure 3 for details.
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The above figure is
reproduced from the cited reference
with permission from the Protein Society
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Secondary reference #3
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Title
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Mechanism of cyanogenesis: the crystal structure of hydroxynitrile lyase from hevea brasiliensis.
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Authors
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U.G.Wagner,
M.Hasslacher,
H.Griengl,
H.Schwab,
C.Kratky.
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Ref.
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Structure, 1996,
4,
811-822.
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PubMed id
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