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PDBsum entry 1qq9
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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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Interactions of streptomyces griseus aminopeptidase with a methionine product analogue: a structural study at 1.53 a resolution.
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Authors
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R.Gilboa,
H.M.Greenblatt,
M.Perach,
A.Spungin-Bialik,
U.Lessel,
G.Wohlfahrt,
D.Schomburg,
S.Blumberg,
G.Shoham.
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Ref.
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Acta Crystallogr D Biol Crystallogr, 2000,
56,
551-558.
[DOI no: ]
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PubMed id
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Abstract
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SGAP is an aminopeptidase present in the extracellular fluid of Streptomyces
griseus cultures. It is a double-zinc enzyme with a strong preference for large
hydrophobic amino-terminus residues. It is a monomeric (30 kDa) heat-stable
enzyme, with a high and efficient catalytic activity modulated by calcium ions.
The small size, high activity and heat stability make SGAP a very attractive
enzyme for various biotechnological applications. Only one other related
aminopeptidase (Aeromonas proteolytica AP; AAP) has been structurally analyzed
to date and its structure was shown to be considerably similar to SGAP, despite
the low sequence homology between the two enzymes. The motivation for the
detailed structural analysis of SGAP originated from a strong mechanistic
interest in the family of double-zinc aminopeptidases, combined with the high
potential applicability of these enzymes. The 1.75 A crystallographic structure
of native SGAP has been previously reported, but did not allow critical
mechanistic interpretations owing to inconclusive structural regions around the
active site. A more accurate structure of SGAP at 1.58 A resolution is reported
in this paper, along with the 1.53 A resolution structure of the SGAP complex
with inhibitory methionine, which is also a product of the SGAP catalytic
process. These two high-resolution structures enable a better understanding of
the SGAP binding mode of both substrates and products. These studies allowed the
tracing of the previously disordered region of the enzyme (Glu196-Arg202) and
the identification of some of the functional groups of the enzyme that are
involved in enzyme-substrate interactions (Asp160, Met161, Gly201, Arg202 and
Phe219). These studies also suggest that Glu131 is directly involved in the
catalytic mechanism of SGAP, probably as the hydrolytic nucleophile. The
structural results are compared with a recent structure of AAP with an
hydroxamate inhibitor in order to draw general functional conclusions which are
relevant for this family of low molecular-weight aminopeptidases.
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Figure 3.
Figure 3 Stereoview of the SGAP active site. The refined new
structure of the native enzyme at 1.58 Å (blue) is superimposed
on the old structure of the native enzyme at 1.75 Å (green). It
is demonstrated that most of the active site is identical,
except for the non-protein ligand of the active-site zinc ions
[a phosphate ion (PO4) for the old structure and a water
molecule (WAT) for the new structure] observed approximately in
the same coordination site between the two metal ions (Zn1 and
Zn2). The difference in this zinc ligand is probably the main
factor causing positional differences for the active-site water
molecules between the old (green) and the new (blue) structures.
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Figure 6.
Figure 6 Interactions of the bound methionine with SGAP as
demonstrated in a schematic diagram of the active-site region of
the SGAP-Met complex. The protein bonds are shown in blue, the
bound methionine (Met) bonds are shown in orange, the Zn atoms
are shown in purple, while the rest of the atoms are in the
standard atomic colors. Dashed lines indicate hydrogen bonds or
ionic interactions, while `radiating' spheres indicate
hydrophobic contacts between the bound methionine (small
spheres) and the neighbouring protein groups (larger spheres).
[This figure was prepared with the program Ligplot (Wallace et
al., 1995[Wallace, A. C., Laskowski, R. A. & Thornton, J. M.
(1995). Protein Eng. 8, 127-134.]).]
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The above figures are
reprinted
by permission from the IUCr:
Acta Crystallogr D Biol Crystallogr
(2000,
56,
551-558)
copyright 2000.
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Secondary reference #1
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Title
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Inhibition of streptomyces griseus aminopeptidase and effects of calcium ions on catalysis and binding--Comparisons with the homologous enzyme aeromonas proteolytica aminopeptidase.
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Authors
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G.Papir,
A.Spungin-Bialik,
D.Ben-Meir,
E.Fudim,
R.Gilboa,
H.M.Greenblatt,
G.Shoham,
U.Lessel,
D.Schomburg,
R.Ashkenazi,
S.Blumberg.
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Ref.
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Eur J Biochem, 1998,
258,
313-319.
[DOI no: ]
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PubMed id
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Figure 2.
Fig. 2. Structure of S. griseus AP inhibitors.
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Figure 5.
Fig. 5. Schematic ribbon diagram of native S. griseus AP showing a `hydrogen bonding network' connecting the Ca
2+
binding site (top, Ca
2+
shown as purple ball) and the active site (bottom, Zn
2+
shown as orange balls). The amino acid side chains participating in this network and
those liganded to the Ca
21
and Zn
21
are shown in yellow (stick representation), while the relevant hydrogen bonds are presented by thin black lines.
This network includes Asp262 (one of the Ca
21
ligands), Asp258, Asn257, Gly163, Asn96 and Asp97 (one of the Zn
21
ligands).
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The above figures are
reproduced from the cited reference
with permission from the Federation of European Biochemical Societies
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Secondary reference #2
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Title
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Streptomyces griseus aminopeptidase: X-Ray crystallographic structure at 1.75 a resolution.
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Authors
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H.M.Greenblatt,
O.Almog,
B.Maras,
A.Spungin-Bialik,
D.Barra,
S.Blumberg,
G.Shoham.
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Ref.
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J Mol Biol, 1997,
265,
620-636.
[DOI no: ]
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PubMed id
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Figure 5.
Figure 5. Stereo view of the initial difference electron
density in the calcium binding site of SGAP. Continuous
contours are at 5.0s, broken contours are at -3.2s.
Structure shown is that of the starting model (taken from
the refined model of the apo-enzyme), which was used
to calculate the difference map shown here. All residues
which contribute ligands to the calcium ion are labelled,
as well as the water molecule (OW) replaced by the
cation.
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Figure 6.
Figure 6. The environment of Met161 in native SGAP.
Stereo view of the electron density present near Met161
S
d
, after ten cycles of refinement. Contouring of the
(Fo - Fc ) map is at 4.5s, with a cover radius of 1.5 Å . The
protein model shown, with the extra oxygen atom
labelled (O), is the final refined structure. The two zinc
cations are shown for reference.
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The above figures are
reproduced from the cited reference
with permission from Elsevier
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Secondary reference #3
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Title
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Aminopeptidase from streptomyces griseus: primary structure and comparison with other zinc-Containing aminopeptidases.
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Authors
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B.Maras,
H.M.Greenblatt,
G.Shoham,
A.Spungin-Bialik,
S.Blumberg,
D.Barra.
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Ref.
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Eur J Biochem, 1996,
236,
843-846.
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PubMed id
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