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Title
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Site-directed mutagenesis of the catalytic tryptophan environment in Pleurotus eryngii versatile peroxidase.
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Authors
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F.J.Ruiz-Dueñas,
M.Morales,
M.J.Mate,
A.Romero,
M.J.Martínez,
A.T.Smith,
A.T.Martínez.
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Ref.
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Biochemistry, 2008,
47,
1685-1695.
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PubMed id
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Abstract
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Lignin degradation by fungal peroxidases is initiated by one-electron transfer
to an exposed tryptophan radical, a reaction mediated by veratryl alcohol (VA)
in lignin peroxidase (LiP). Versatile peroxidase (VP) differs not only in its
oxidation of Mn2+ at a second catalytic site but also in its ability to directly
oxidize different aromatic compounds. The catalytic tryptophan environment was
compared in LiP and VP crystal structures, and six residues near VP Trp164 were
modified by site-directed mutagenesis. Oxidation of Mn2+ was practically
unaffected. However, several mutations modified the oxidation kinetics of the
high-redox-potential substrates VA and Reactive Black 5 (RB5), demonstrating
that other residues contribute to substrate oxidation by the Trp164 radical.
Introducing acidic residues at the tryptophan environment did not increase the
efficiency of VP oxidizing VA. On the contrary, all variants harboring the R257D
mutation lost their activity on RB5. Interestingly, this activity was restored
when VA was added as a mediator, revealing the LiP-type behavior of this
variant. Moreover, combination of the A260F and R257A mutations strongly
increased (20-50-fold) the apparent second-order rate constants for reduction of
VP compounds I and II by VA to values similar to those found in LiP.
Dissociation of the enzyme-product complex seemed to be the limiting step in the
turnover of this improved variant. Nonexposed residues in the vicinity of Trp164
can also affect VP activity, as found with the M247F mutation. This was a direct
effect since no modification of the surrounding residues was found in the
crystal structure of this variant.
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