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PDBsum entry 1txr
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* Residue conservation analysis
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Enzyme class:
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E.C.3.4.11.10
- bacterial leucyl aminopeptidase.
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Reaction:
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Release of an N-terminal amino acid, preferentially leucine, but not glutamic or aspartic acids.
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Cofactor:
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Zn(2+)
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DOI no:
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Biochemistry
43:9620-9628
(2004)
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PubMed id:
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Spectroscopic and X-ray crystallographic characterization of bestatin bound to the aminopeptidase from Aeromonas (Vibrio) proteolytica.
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C.C.Stamper,
D.L.Bienvenue,
B.Bennett,
D.Ringe,
G.A.Petsko,
R.C.Holz.
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ABSTRACT
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Binding of the competitive, slow-binding inhibitor bestatin
([(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoy]-leucine) to the aminopeptidase from
Aeromonas proteolytica (AAP) was examined by both spectroscopic and
crystallographic methods. Electronic absorption spectra of the catalytically
competent [Co_(AAP)], [CoCo(AAP)], and [ZnCo(AAP)] enzymes recorded in the
presence of bestatin revealed that both of the divalent metal ions in AAP are
involved in binding bestatin. The electron paramagnetic resonance (EPR) spectrum
of the [CoCo(AAP)]-bestatin complex exhibited no observable perpendicular- or
parallel-mode signal. These data indicate that the two Co(II) ions in AAP are
antiferromagnetically coupled yielding an S = 0 ground state and suggest that a
single oxygen atom bridges between the two divalent metal ions. The EPR data
obtained for [CoZn(AAP)] and [ZnCo(AAP)] confirm that bestatin interacts with
both metal ions. The X-ray crystal structure of the [ZnZn(AAP)]-bestatin complex
was solved to 2.0 A resolution. Both side chains of bestatin occupy a
well-defined hydrophobic pocket that is adjacent to the dinuclear Zn(II) active
site. The amino acid residues ligated to the dizinc(II) cluster in AAP are
identical to those in the native structure with only minor perturbations in bond
length. The alkoxide oxygen of bestatin bridges between the two Zn(II) ions in
the active site, displacing the bridging water molecule observed in the native
[ZnZn(AAP)] structure. The M-M distances observed in the AAP-bestatin complex
and native AAP are identical (3.5 A) with alkoxide oxygen atom distances of 2.1
and 1.9 A from Zn1 and Zn2, respectively. Interestingly, the backbone carbonyl
oxygen atom of bestatin is coordinated to Znl at a distance of 2.3 A. In
addition, the NH(2) group of bestatin, which mimics the N-terminal amine group
of an incoming peptide, binds to Zn2 with a bond distance of 2.3 A. A
combination of the spectroscopic and X-ray crystallographic data presented
herein with the previously reported mechanistic data for AAP has provided
additional insight into the substrate-binding step of peptide hydrolysis as well
as insight into important small molecule features for inhibitor design.
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Literature references that cite this PDB file's key reference
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PubMed id
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Reference
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H.Y.Yoon,
S.H.Shim,
L.J.Baek,
and
J.I.Hong
(2011).
Small-molecule probe using dual signals to monitor leucine aminopeptidase activity.
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Bioorg Med Chem Lett,
21,
2403-2405.
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B.P.Nocek,
D.M.Gillner,
Y.Fan,
R.C.Holz,
and
A.Joachimiak
(2010).
Structural basis for catalysis by the mono- and dimetalated forms of the dapE-encoded N-succinyl-L,L-diaminopimelic acid desuccinylase.
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J Mol Biol,
397,
617-626.
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PDB codes:
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H.Huang,
H.Tanaka,
B.D.Hammock,
and
C.Morisseau
(2009).
Novel and highly sensitive fluorescent assay for leucine aminopeptidases.
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Anal Biochem,
391,
11-16.
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M.Hartley,
W.Yong,
and
B.Bennett
(2009).
Heterologous expression and purification of Vibrio proteolyticus (Aeromonas proteolytica) aminopeptidase: a rapid protocol.
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Protein Expr Purif,
66,
91.
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PDB code:
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S.Mitra,
G.Sheppard,
J.Wang,
B.Bennett,
and
R.C.Holz
(2009).
Analyzing the binding of Co(II)-specific inhibitors to the methionyl aminopeptidases from Escherichia coli and Pyrococcus furiosus.
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J Biol Inorg Chem,
14,
573-585.
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H.Unno,
T.Yamashita,
S.Ujita,
N.Okumura,
H.Otani,
A.Okumura,
K.Nagai,
and
M.Kusunoki
(2008).
Structural Basis for Substrate Recognition and Hydrolysis by Mouse Carnosinase CN2.
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J Biol Chem,
283,
27289-27299.
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PDB codes:
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B.M.McArdle,
and
R.J.Quinn
(2007).
Identification of protein fold topology shared between different folds inhibited by natural products.
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Chembiochem,
8,
788-798.
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W.C.McGregor,
S.I.Swierczek,
B.Bennett,
and
R.C.Holz
(2007).
Characterization of the catalytically active Mn(II)-loaded argE-encoded N-acetyl-L-ornithine deacetylase from Escherichia coli.
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J Biol Inorg Chem,
12,
603-613.
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J.Arima,
Y.Uesugi,
M.Uraji,
S.Yatsushiro,
S.Tsuboi,
M.Iwabuchi,
and
T.Hatanaka
(2006).
Modulation of Streptomyces leucine aminopeptidase by calcium: identification and functional analysis of key residues in activation and stabilization by calcium.
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J Biol Chem,
281,
5885-5894.
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Q.Z.Ye,
S.X.Xie,
Z.Q.Ma,
M.Huang,
and
R.P.Hanzlik
(2006).
Structural basis of catalysis by monometalated methionine aminopeptidase.
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Proc Natl Acad Sci U S A,
103,
9470-9475.
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PDB codes:
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The most recent references are shown first.
Citation data come partly from CiteXplore and partly
from an automated harvesting procedure. Note that this is likely to be
only a partial list as not all journals are covered by
either method. However, we are continually building up the citation data
so more and more references will be included with time.
Where a reference describes a PDB structure, the PDB
codes are
shown on the right.
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Links
