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PDBsum entry 2rl3
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References listed in PDB file
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Key reference
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Title
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Critical role of tryptophan 154 for the activity and stability of class d beta-Lactamases.
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Authors
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S.Baurin,
L.Vercheval,
F.Bouillenne,
C.Falzone,
A.Brans,
L.Jacquamet,
J.L.Ferrer,
E.Sauvage,
D.Dehareng,
J.M.Frère,
P.Charlier,
M.Galleni,
F.Kerff.
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Ref.
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Biochemistry, 2009,
48,
11252-11263.
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PubMed id
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Abstract
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The catalytic efficiency of the class D beta-lactamase OXA-10 depends critically
on an unusual carboxylated lysine as the general base residue for both the
enzyme acylation and deacylation steps of catalysis. Evidence is presented that
the interaction between the indole group of Trp154 and the carboxylated lysine
is essential for the stability of the posttranslationally modified Lys70.
Substitution of Trp154 by Gly, Ala, or Phe yielded noncarboxylated enzymes which
displayed poor catalytic efficiencies and reduced stability when compared to the
wild-type OXA-10. The W154H mutant was partially carboxylated. In addition, the
maximum values of k(cat) and k(cat)/K(M) were shifted toward pH 7, indicating
that the carboxylation state of Lys70 is dependent on the protonation level of
the histidine. A comparison of the three-dimensional structures of the different
proteins also indicated that the Trp154 mutations did not modify the overall
structures of OXA-10 but induced an increased flexibility of the Omega-loop in
the active site. Finally, the deacylation-impaired W154A mutant was used to
determine the structure of the acyl-enzyme complex with benzylpenicillin. These
results indicate a role of the Lys70 carboxylation during the deacylation step
and emphasize the importance of Trp154 for the ideal positioning of active site
residues leading to an optimum activity.
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