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PDBsum entry 2wsd
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Oxidoreductase
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PDB id
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2wsd
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References listed in PDB file
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Key reference
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Title
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Proximal mutations at the type 1 copper site of cota laccase: spectroscopic, Redox, Kinetic and structural characterization of i494a and l386a mutants.
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Authors
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P.Durão,
Z.Chen,
C.S.Silva,
C.M.Soares,
M.M.Pereira,
S.Todorovic,
P.Hildebrandt,
I.Bento,
P.F.Lindley,
L.O.Martins.
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Ref.
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Biochem J, 2008,
412,
339-346.
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PubMed id
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Abstract
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In the present study the CotA laccase from Bacillus subtilis has been mutated at
two hydrophobic residues in the vicinity of the type 1 copper site. The mutation
of Leu(386) to an alanine residue appears to cause only very subtle alterations
in the properties of the enzyme indicating minimal changes in the structure of
the copper centres. However, the replacement of Ile(494) by an alanine residue
leads to significant changes in the enzyme. Thus the major visible absorption
band is upshifted by 16 nm to 625 nm and exhibits an increased intensity,
whereas the intensity of the shoulder at approx. 330 nm is decreased by a factor
of two. Simulation of the EPR spectrum of the I494A mutant reveals differences
in the type 1 as well as in the type 2 copper centre reflecting modifications of
the geometry of these centres. The intensity weighted frequencies
<nu(Cu-S)>, calculated from resonance Raman spectra are 410 cm(-1) for the
wild-type enzyme and 396 cm(-1) for the I494A mutant, indicating an increase of
the Cu-S bond length in the type 1 copper site of the mutant. Overall the data
clearly indicate that the Ile(494) mutation causes a major alteration of the
structure near the type 1 copper site and this has been confirmed by X-ray
crystallography. The crystal structure shows the presence of a fifth ligand, a
solvent molecule, at the type 1 copper site leading to an approximate trigonal
bipyramidal geometry. The redox potentials of the L386A and I494A mutants are
shifted downwards by approx. 60 and 100 mV respectively. These changes correlate
well with decreased catalytic efficiency of both mutants compared with the
wild-type.
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