 |
PDBsum entry 1rk5
|
|
|
|
|
 |
Contents |
 |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
* Residue conservation analysis
|
|
|
|
|
References listed in PDB file
|
|
|
Key reference
|
|
|
Title
|
|
The functional role of the binuclear metal center in d-Aminoacylase: one-Metal activation and second-Metal attenuation.
|
|
|
Authors
|
|
W.L.Lai,
L.Y.Chou,
C.Y.Ting,
R.Kirby,
Y.C.Tsai,
A.H.Wang,
S.H.Liaw.
|
|
|
Ref.
|
|
J Biol Chem, 2004,
279,
13962-13967.
[DOI no: ]
|
|
|
PubMed id
|
|
|
|
|
|
Abstract
|
|
|
Our structural comparison of the TIM barrel metal-dependent hydrolase(-like)
superfamily suggests a classification of their divergent active sites into four
types: alphabeta-binuclear, alpha-mononuclear, beta-mononuclear, and
metal-independent subsets. The d-aminoacylase from Alcaligenes faecalis DA1
belongs to the beta-mononuclear subset due to the fact that the catalytically
essential Zn(2+) is tightly bound at the beta site with coordination by Cys(96),
His(220), and His(250), even though it possesses a binuclear active site with a
weak alpha binding site. Additional Zn(2+), Cd(2+), and Cu(2+), but not Ni(2+),
Co(2+), Mg(2+), Mn(2+), and Ca(2+), can inhibit enzyme activity. Crystal
structures of these metal derivatives show that Zn(2+) and Cd(2+) bind at the
alpha(1) subsite ligated by His(67), His(69), and Asp(366), while Cu(2+) at the
alpha(2) subsite is chelated by His(67), His(69) and Cys(96). Unexpectedly, the
crystal structure of the inactive H220A mutant displays that the endogenous
Zn(2+) shifts to the alpha(3) subsite coordinated by His(67), His(69), Cys(96),
and Asp(366), revealing that elimination of the beta site changes the
coordination geometry of the alpha ion with an enhanced affinity. Kinetic
studies of the metal ligand mutants such as C96D indicate the uniqueness of the
unusual bridging cysteine and its involvement in catalysis. Therefore, the two
metal-binding sites in the d-aminoacylase are interactive with partially mutual
exclusion, thus resulting in widely different affinities for the
activation/attenuation mechanism, in which the enzyme is activated by the metal
ion at the beta site, but inhibited by the subsequent binding of the second ion
at the alpha site.
|
|
|
|
|
|
|
|
Figure 3.
FIG. 3. The metal centers. A, the F[o] - F[c] electron
density maps of the native enzyme in complex with 100 mM ZnCl[2]
contoured at 15  level and shown in
magenta, with 50 mM CdCl[2] contoured at 15 level and shown in
cyan, and with 100 mM CuCl[2] contoured at 18 level
and shown in green. The metal ligands are shown as a
ball-and-stick representation, with the Zn2+ and Cu2+ ions as
magenta and green spheres, respectively. Zn2+ and Cd^2+ bind at
the  subsite, where Cu2+
binds at the [2] subsite. B, the
2F[o] - F[c] electron density maps of the H220A mutant contoured
at 2.5 level and shown in
cyan, and the difference map for the zinc ion contoured at 15
level and shown in
magenta. The endogenous zinc ion binds at the [3]
subsite instead of the  site in this mutant. C,
the 2F[o] - F[c] electron density map of the D366A mutant
contoured at 2.5 level and shown in
cyan, and the difference map for the zinc ion in complex with
100 mM ZnCl[2] contoured at 15 level and shown in
magenta. The additional zinc ion binds at the [4]
subsite. D, superposition of the native enzyme with 100 mM
ZnCl[2] in blue, the native enzyme with 100 CuCl[2] in green,
the H220A mutant in yellow, and the D366A mutant with 100 mM
ZnCl[2] in red. The different metal coordination is carried out
by small shifts in the side chains of ligands and small
movements of the metal ions.
|
|
Figure 4.
FIG. 4. The proposed mechanisms for catalysis (A) and metal
attenuation (B). The numbers shown indicate the interatomic
distances in angstroms. Asp366 maybe with assistance from His67
and His69, is responsible for the proton transfer from the water
molecule to the amide nitrogen (3). The presence of the
inhibitory metal ion at the [1] site might lower
the pK[a] values of its ligand residues, His67, His69, and
Asp366, and/or hold the active site water to perturb the proton
shuttle and intermediate stabilization.
|
|
|
|
|
|
The above figures are
reprinted
by permission from the ASBMB:
J Biol Chem
(2004,
279,
13962-13967)
copyright 2004.
|
|
|
Secondary reference #1
|
|
|
Title
|
|
Crystal structure of d-Aminoacylase from alcaligenes faecalis da1. A novel subset of amidohydrolases and insights into the enzyme mechanism.
|
|
|
Authors
|
|
S.H.Liaw,
S.J.Chen,
T.P.Ko,
C.S.Hsu,
C.J.Chen,
A.H.Wang,
Y.C.Tsai.
|
|
|
Ref.
|
|
J Biol Chem, 2003,
278,
4957-4962.
[DOI no: ]
|
|
|
PubMed id
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Figure 3.
Fig. 3. The metal center. A, the 2F[o]  F[c]
electron density map in the zinc center contoured at the 3  level and
is shown in green, and the weak density in the 2F[o] F[c] map
for the loosely bound zinc ion contoured at the 2 level and
is shown in purple. The structural refinement revealed that the
enzyme binds two zinc ions with very different affinities. B,
superposition of the bi-nickel center in urease (Protein Data
Bank code 1UBP), the mononuclear iron center in cytosine
deaminase (Protein Data Bank code 1K6W), and the bi-zinc center
in D-aminoacylase, shown in red, blue, and green, respectively.
The 
metal-binding site is more buried, while the  site is
more solvent-exposed. The residue numbering is labeled in the
same color for each protein. The critical hallmark for the
binuclear subset is a carboxylated lysine residue serving as a
bridging ligand. A cysteine residue (Cys96) in D-aminoacylse,
and the third conserved histidine (His214) in cytosine
deaminase, compensate the missing carboxylated lysine.
|
|
Figure 4.
Fig. 4. The putative substrate-binding site. A, the two
acetate-binding sites in the active site cavity (Act1 and Act2).
Residues surrounded the binding site are displayed as
ball-and-stick representations and the zinc ion as a purple
sphere. B, the proposed substrate-binding site with the modeled
N-acetyl-D-methionine in ball-and-stick and the active water
molecule (Wat) as a green sphere. The oxygen atoms of the
acetates and substrate are expected to bind at the same position
because of the extensive interactions. The first acetate may
displace the attacking water molecule at the metal center.
|
|
|
|
|
|
The above figures are
reproduced from the cited reference
with permission from the ASBMB
|
|
|
Secondary reference #2
|
|
|
Title
|
|
Structural-Based mutational analysis of d-Aminoacylase from alcaligenes faecalis da1.
|
|
|
Authors
|
|
C.S.Hsu,
W.L.Lai,
W.W.Chang,
S.H.Liaw,
Y.C.Tsai.
|
|
|
Ref.
|
|
Protein Sci, 2002,
11,
2545-2550.
[DOI no: ]
|
|
|
PubMed id
|
|
|
|
|
|
|
|
|
|
|
|
Figure 3.
Fig. 3. The proposed bi-zinc center in the DA1
D-aminoacylases on the basis of structural prediction and
mutational studies.
|
|
|
|
|
|
The above figure is
reproduced from the cited reference
with permission from the Protein Society
|
|
|
|
|
|
|
