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PDBsum entry 1djc
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References listed in PDB file
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Key reference
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Title
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Structure and kinetics of the beta-Lactamase mutants s70a and k73h from staphylococcus aureus pc1.
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Authors
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C.C.Chen,
T.J.Smith,
G.Kapadia,
S.Wäsch,
L.E.Zawadzke,
A.Coulson,
O.Herzberg.
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Ref.
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Biochemistry, 1996,
35,
12251-12258.
[DOI no: ]
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PubMed id
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Abstract
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Two mutant beta-lactamases from Staphylococcus aureus PC1 which probe key
catalytic residues have been produced by site-directed mutagenesis. In the S70A
enzyme, the nucleophilic group that attacks the beta-lactam carbonyl carbon atom
was eliminated. Consequently, the kcat values for hydrolysis of benzylpenicillin
and nitrocefin have been reduced by 10(4)-10(5) compared with the wild-type
enzyme. The crystal structure of S70A beta-lactamase has been determined at 2.1
A resolution. With the exception of the mutation site, the structure is
identical to that of the native enzyme. The residual activity is attributed
either to mistranslation that leads to production of wild-type enzyme and/or to
remaining features of the active site that stabilize the tetrahedral transition
state. Soaking of the crystals with ampicillin or clavulanate, followed by
flash-freezing, has been carried out and the structures examined at 2.0 A
resolution. For both experiments, the difference electron density maps revealed
buildup of density in the active site that presumably corresponds to beta-lactam
binding. However, neither electron density is sufficiently clear for defining
the atomic details of the bound compounds. The K73H beta-lactamase has been
prepared to test the possible role of Lys73 in proton transfer. It exhibits no
detectable activity toward benzylpenicillin, and 10(5)-fold reduction of kcat
for nitrocefin hydrolysis compared with the wild-type enzyme. No significant
recovery of activity has been measured when the pH was varied between 5.0 and
8.0. The crystal structure of K73H beta-lactamase has been determined at 1.9 A
resolution. While the overall structure is similar to that of the native enzyme,
the electrostatic interactions between His73 and neighboring residues indicate
that the imidazole ring is positively charged. In addition, the hydroxyl group
of Ser70 adopts a position that is incompatible with nucleophilic attack on
substrates. A crystal soaked with ampicillin was flash-frozen, and diffraction
data were collected at 2.1 A resolution. The electron density map showed no
indication of substrate binding.
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Secondary reference #1
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Title
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An engineered staphylococcus aureus pc1 beta-Lactamase that hydrolyses third-Generation cephalosporins.
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Authors
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L.E.Zawadzke,
T.J.Smith,
O.Herzberg.
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Ref.
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Protein Eng, 1995,
8,
1275-1285.
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PubMed id
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Secondary reference #2
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Title
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Inhibition of beta-Lactamase by clavulanate. Trapped intermediates in cryocrystallographic studies.
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Authors
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C.C.Chen,
O.Herzberg.
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Ref.
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J Mol Biol, 1992,
224,
1103-1113.
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PubMed id
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Secondary reference #3
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Title
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Refined crystal structure of beta-Lactamase from staphylococcus aureus pc1 at 2.0 a resolution.
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Author
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O.Herzberg.
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Ref.
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J Mol Biol, 1991,
217,
701-719.
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PubMed id
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Secondary reference #4
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Title
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Bacterial resistance to beta-Lactam antibiotics: crystal structure of beta-Lactamase from staphylococcus aureus pc1 at 2.5 a resolution.
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Authors
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O.Herzberg,
J.Moult.
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Ref.
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Science, 1987,
236,
694-701.
[DOI no: ]
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PubMed id
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