 |
PDBsum entry 1c0p
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
 |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Oxidoreductase
|
PDB id
|
|
|
|
1c0p
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
 |
Contents |
 |
|
|
|
|
|
|
|
|
|
|
|
|
* Residue conservation analysis
|
|
|
|
|
References listed in PDB file
|
|
|
Key reference
|
|
|
Title
|
|
The X-Ray structure of d-Amino acid oxidase at very high resolution identifies the chemical mechanism of flavin-Dependent substrate dehydrogenation.
|
|
|
Authors
|
|
S.Umhau,
L.Pollegioni,
G.Molla,
K.Diederichs,
W.Welte,
M.S.Pilone,
S.Ghisla.
|
|
|
Ref.
|
|
Proc Natl Acad Sci U S A, 2000,
97,
12463-12468.
[DOI no: ]
|
|
|
PubMed id
|
|
|
|
|
|
Abstract
|
|
|
Flavin is one of the most versatile redox cofactors in nature and is used by
many enzymes to perform a multitude of chemical reactions. d-Amino acid oxidase
(DAAO), a member of the flavoprotein oxidase family, is regarded as a key enzyme
for the understanding of the mechanism underlying flavin catalysis. The very
high-resolution structures of yeast DAAO complexed with d-alanine,
d-trifluoroalanine, and l-lactate (1.20, 1.47, and 1.72 A) provide strong
evidence for hydride transfer as the mechanism of dehydrogenation. This is
inconsistent with the alternative carbanion mechanism originally favored for
this type of enzymatic reaction. The step of hydride transfer can proceed
without involvement of amino acid functional groups. These structures, together
with results from site-directed mutagenesis, point to orbital
orientation/steering as the major factor in catalysis. A diatomic species,
proposed to be a peroxide, is found at the active center and on the Re-side of
the flavin. These results are of general relevance for the mechanisms of
flavoproteins and lead to the proposal of a common dehydrogenation mechanism for
oxidases and dehydrogenases.
|
|
|
|
|
|
|
|
Figure 3.
Fig. 3. Active site of RgDAAO at 1.2-Å resolution.
Stereo view of the 2F[obs]  F[calc]
map (orange, 3  ) and the
omit map (magenta, 3 ) showing
clear electron density assigned to a peroxide species. The data
were obtained from RgDAAO crystals soaked with 20 mM D-alanine
and 200 mM pyruvate.
|
|
Figure 4.
Fig. 4. Reciprocal orientation of ligands and flavin
plane. For clarity, the dioxygen species has been omitted.
Dashed lines represent H-bonds. (A) D-Ala is viewed along its N-
 C axis; the
electron density is shown at 2 . (B)
D-F[3]-Ala (Upper) and L-lactate (Lower). The green trace
represents the ideal line connecting the flavin N(5) and the
ligand C centers
(distance  3.2 Å).
Note that the C---H
function (grey) of L-lactate points away from the flavin. With
L-lactate, the position of the ---H
results from H-inclusion in the refinement. In the case of
D-F[3]-Ala (A), the number of observations (1.72 Å) does
not allow positioning. The strong H-bond interactions with the
-NH[2]/OH,
together with the electrostatic interaction of the substrate
carboxylate group with Arg-285, Tyr-238, and Tyr-223, provide
the rationale for substrate D-specificity in that they prevent
binding of the L-amino acid in a productive manner.
|
|
|
|
|
|
|
|
|
|
