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PDBsum entry 2flt
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References listed in PDB file
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Key reference
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Title
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Crystal structures of native and inactivated cis-3-Chloroacrylic acid dehalogenase. Structural basis for substrate specificity and inactivation by (r)-Oxirane-2-Carboxylate.
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Authors
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R.M.De jong,
P.Bazzacco,
G.J.Poelarends,
W.H.Johnson,
Y.J.Kim,
E.A.Burks,
H.Serrano,
A.M.Thunnissen,
C.P.Whitman,
B.W.Dijkstra.
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Ref.
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J Biol Chem, 2007,
282,
2440-2449.
[DOI no: ]
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PubMed id
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Abstract
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The bacterial degradation pathways for the nematocide 1,3-dichloropropene rely
on hydrolytic dehalogenation reactions catalyzed by cis- and
trans-3-chloroacrylic acid dehalogenases (cis-CaaD and CaaD, respectively).
X-ray crystal structures of native cis-CaaD and cis-CaaD inactivated by
(R)-oxirane-2-carboxylate were elucidated. They locate four known catalytic
residues (Pro-1, Arg-70, Arg-73, and Glu-114) and two previously unknown,
potential catalytic residues (His-28 and Tyr-103'). The Y103F and H28A mutants
of these latter two residues displayed reductions in cis-CaaD activity
confirming their importance in catalysis. The structure of the inactivated
enzyme shows covalent modification of the Pro-1 nitrogen atom by
(R)-2-hydroxypropanoate at the C3 position. The interactions in the complex
implicate Arg-70 or a water molecule bound to Arg-70 as the proton donor for the
epoxide ring-opening reaction and Arg-73 and His-28 as primary binding contacts
for the carboxylate group. This proposed binding mode places the (R)-enantiomer,
but not the (S)-enantiomer, in position to covalently modify Pro-1. The absence
of His-28 (or an equivalent) in CaaD could account for the fact that CaaD is not
inactivated by either enantiomer. The cis-CaaD structures support a mechanism in
which Glu-114 and Tyr-103' activate a water molecule for addition to C3 of the
substrate and His-28, Arg-70, and Arg-73 interact with the C1 carboxylate group
to assist in substrate binding and polarization. Pro-1 provides a proton at C2.
The involvement of His-28 and Tyr-103' distinguishes the cis-CaaD mechanism from
the otherwise parallel CaaD mechanism. The two mechanisms probably evolved
independently as the result of an early gene duplication of a common ancestor.
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Figure 1.
FIGURE 1. Stereo views of (A) the monomeric and (B) the
trimeric structure of cis-CaaD. The catalytic Pro-1 is shown in
ball-and-stick (A) and Corey-Pauling-Koltun (B) representation.
The figures were made using MOLSCRIPT and RASTER3D (24, 25).
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Figure 2.
FIGURE 2. Detailed overview of the structure of native
cis-CaaD and the final electron density of the covalent adduct
in the structure of inactivated cis-CaaD. A, close-up stereo
view of the active site of the native enzyme showing the
interactions between the phosphate/sulfate ion and the two
arginines (Arg-70 and Arg-73) and histidine (His-28). Residues
are labeled by their chain color, except Pro-1, which is labeled
in black. B, stereo view of the final 2F[o] – F[c]
electron-density map contoured at 1.0  from XtalView (20)
covering the proline and the covalently bound
(R)-2-hydroxypropanoate adduct, clearly showing the tetrahedral
conformations at the prolyl nitrogen and C2 of the adduct. The
figures were prepared with MOLSCRIPT (A) (24) and RASTER3D (A
and B) (24, 25).
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The above figures are
reprinted
by permission from the ASBMB:
J Biol Chem
(2007,
282,
2440-2449)
copyright 2007.
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