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* Residue conservation analysis
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Enzyme class:
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Chains A, B:
E.C.3.5.1.11
- penicillin amidase.
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Pathway:
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Penicillin Biosynthesis and Metabolism
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Reaction:
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a penicillin + H2O = 6-aminopenicillanate + a carboxylate
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penicillin
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+
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H2O
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=
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6-aminopenicillanate
Bound ligand (Het Group name = )
matches with 41.18% similarity
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+
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carboxylate
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Molecule diagrams generated from .mol files obtained from the
KEGG ftp site
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Protein Eng
13:857-863
(2000)
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PubMed id:
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Characterization of the beta-lactam binding site of penicillin acylase of Escherichia coli by structural and site-directed mutagenesis studies.
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W.B.Alkema,
C.M.Hensgens,
E.H.Kroezinga,
E.de Vries,
R.Floris,
J.M.van der Laan,
B.W.Dijkstra,
D.B.Janssen.
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ABSTRACT
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The binding of penicillin to penicillin acylase was studied by X-ray
crystallography. The structure of the enzyme-substrate complex was determined
after soaking crystals of an inactive betaN241A penicillin acylase mutant with
penicillin G. Binding of the substrate induces a conformational change, in which
the side chains of alphaF146 and alphaR145 move away from the active site, which
allows the enzyme to accommodate penicillin G. In the resulting structure, the
beta-lactam binding site is formed by the side chains of alphaF146 and betaF71,
which have van der Waals interactions with the thiazolidine ring of penicillin G
and the side chain of alphaR145 that is connected to the carboxylate group of
the ligand by means of hydrogen bonding via two water molecules. The backbone
oxygen of betaQ23 forms a hydrogen bond with the carbonyl oxygen of the
phenylacetic acid moiety through a bridging water molecule. Kinetic studies
revealed that the site-directed mutants alphaF146Y, alphaF146A and alphaF146L
all show significant changes in their interaction with the beta-lactam
substrates as compared with the wild type. The alphaF146Y mutant had the same
affinity for 6-aminopenicillanic acid as the wild-type enzyme, but was not able
to synthesize penicillin G from phenylacetamide and 6-aminopenicillanic acid.
The alphaF146L and alphaF146A enzymes had a 3-5-fold decreased affinity for
6-aminopenicillanic acid, but synthesized penicillin G more efficiently than the
wild type. The combined results of the structural and kinetic studies show the
importance of alphaF146 in the beta-lactam binding site and provide leads for
engineering mutants with improved synthetic properties.
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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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M.Bokhove,
H.Yoshida,
C.M.Hensgens,
J.M.van der Laan,
J.D.Sutherland,
and
B.W.Dijkstra
(2010).
Structures of an isopenicillin N converting Ntn-hydrolase reveal different catalytic roles for the active site residues of precursor and mature enzyme.
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Structure,
18,
301-308.
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PDB codes:
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M.Bokhove,
P.N.Jimenez,
W.J.Quax,
and
B.W.Dijkstra
(2010).
The quorum-quenching N-acyl homoserine lactone acylase PvdQ is an Ntn-hydrolase with an unusual substrate-binding pocket.
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Proc Natl Acad Sci U S A,
107,
686-691.
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PDB codes:
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D.Zhiryakova,
I.Ivanov,
S.Ilieva,
M.Guncheva,
B.Galunsky,
and
N.Stambolieva
(2009).
Do N-terminal nucleophile hydrolases indeed have a single amino acid catalytic center?
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FEBS J,
276,
2589-2598.
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G.G.Chilov,
O.V.Stroganov,
and
V.K.Svedas
(2008).
Molecular modeling studies of substrate binding by penicillin acylase.
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Biochemistry (Mosc),
73,
56-64.
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D.A.Cecchini,
I.Serra,
D.Ubiali,
M.Terreni,
and
A.M.Albertini
(2007).
New active site oriented glyoxyl-agarose derivatives of Escherichia coli penicillin G acylase.
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BMC Biotechnol,
7,
54.
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G.G.Chilov,
A.V.Sidorova,
and
V.K.Svedas
(2007).
Quantum chemical studies of the catalytic mechanism of N-terminal nucleophile hydrolase.
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Biochemistry (Mosc),
72,
495-500.
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J.Wang,
Q.Zhang,
H.Huang,
Z.Yuan,
D.Ding,
S.Yang,
and
W.Jiang
(2007).
Increasing synthetic performance of penicillin G acylase from Bacillus megaterium by site-directed mutagenesis.
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Appl Microbiol Biotechnol,
74,
1023-1030.
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A.M.Hosie,
S.D.Buckingham,
A.Hamon,
and
D.B.Sattelle
(2006).
Replacement of asparagine with arginine at the extracellular end of the second transmembrane (M2) region of insect GABA receptors increases sensitivity to penicillin G.
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Invert Neurosci,
6,
75-79.
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F.Scaramozzino,
I.Estruch,
P.Rossolillo,
M.Terreni,
and
A.M.Albertini
(2005).
Improvement of catalytic properties of Escherichia coli penicillin G acylase immobilized on glyoxyl agarose by addition of a six-amino-acid tag.
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Appl Environ Microbiol,
71,
8937-8940.
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D.T.Guranda,
T.S.Volovik,
and
V.K.Svedas
(2004).
pH Stability of penicillin acylase from Escherichia coli.
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Biochemistry (Mosc),
69,
1386-1390.
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G.Flores,
X.Soberón,
and
J.Osuna
(2004).
Production of a fully functional, permuted single-chain penicillin G acylase.
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Protein Sci,
13,
1677-1683.
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J.Rajendhran,
and
P.Gunasekaran
(2004).
Recent biotechnological interventions for developing improved penicillin G acylases.
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J Biosci Bioeng,
97,
1.
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M.Guncheva,
I.Ivanov,
B.Galunsky,
N.Stambolieva,
and
J.Kaneti
(2004).
Kinetic studies and molecular modelling attribute a crucial role in the specificity and stereoselectivity of penicillin acylase to the pair ArgA145-ArgB263.
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Eur J Biochem,
271,
2272-2279.
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P.Braiuca,
C.Ebert,
L.Fischer,
L.Gardossi,
and
P.Linda
(2003).
A homology model of penicillin acylase from Alcaligenes faecalis and in silico evaluation of its selectivity.
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Chembiochem,
4,
615-622.
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W.B.Alkema,
E.de Vries,
R.Floris,
and
D.B.Janssen
(2003).
Kinetics of enzyme acylation and deacylation in the penicillin acylase-catalyzed synthesis of beta-lactam antibiotics.
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Eur J Biochem,
270,
3675-3683.
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C.F.Sio,
A.M.Riemens,
J.M.van der Laan,
R.M.Verhaert,
and
W.J.Quax
(2002).
Directed evolution of a glutaryl acylase into an adipyl acylase.
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Eur J Biochem,
269,
4495-4504.
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J.J.Polderman-Tijmes,
P.A.Jekel,
E.J.de Vries,
A.E.van Merode,
R.Floris,
J.M.van der Laan,
T.Sonke,
and
D.B.Janssen
(2002).
Cloning, sequence analysis, and expression in Escherichia coli of the gene encoding an alpha-amino acid ester hydrolase from Acetobacter turbidans.
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Appl Environ Microbiol,
68,
211-218.
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W.B.Alkema,
A.J.Dijkhuis,
E.De Vries,
and
D.B.Janssen
(2002).
The role of hydrophobic active-site residues in substrate specificity and acyl transfer activity of penicillin acylase.
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Eur J Biochem,
269,
2093-2100.
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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
