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In the high-resolution crystal structures of two lignin peroxidase isozymes from
the white rot fungus Phanerochaete chrysosporium a significant electron density
at single bond distance from the C beta of Trp171 was observed and interpreted
as a hydroxy group. To further clarify the nature of this feature, we carried
out tryptic digestion of the enzyme and isolated the Trp171 containing peptide.
Under ambient conditions, this peptide shows an absorbance spectrum typical of
tryptophan. At elevated temperature, however, the formation of an unusual
absorbance spectrum with lambda max = 333 nm can be followed that is identical
to that of N-acetyl-alpha, beta-didehydrotryptophanamide, resulting upon water
elimination from beta-hydroxy tryptophan. The Trp171 containing tryptic peptide
isolated from the recombinant and refolded lignin peroxidase produced from
Escherichia coli does not contain the characteristic 333 nm absorbance band at
any temperature. However, treatment with 3 equiv of H2O2 leads to complete
hydroxylation of Trp171. Reducing substrates compete with this process, e.g., in
the presence of 0.5 mM veratryl alcohol, about 7 equiv of H2O2 is necessary for
complete modification. We conclude that the hydroxylation at the C beta of
Trp171 is an autocatalytic reaction which occurs readily under conditions of
natural turnover, e.g., in the ligninolytic cultures of P. chrysosporium, which
are known to contain an oxidase-based H2O2-generating system. No dependence on
dioxygen was found for this oxidative process. Chemical modification of fungal
lignin peroxidase with the tryptophan-specific agent N-bromo succinimide leads
to a drastically reduced activity with respect to the substrate veratryl
alcohol. This suggests that Trp171 is involved in catalysis and that electron
transfer from this surface residue to the oxidized heme cofactor is possible
under steady-state conditions.
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