Autocatalytic Formation of a Hydroxy Group at Cβ of Trp171 in Lignin Peroxidase

Abstract

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.

A Tryptophan Neutral Radical in the Oxidized State of Versatile Peroxidase from Pleurotus eryngii

“…Similarly, we observed in P. eryngii a tryptophan neutral radical at position 164 (LiP Trp 171 ), which is located at the protein surface. Our results presented in this study strongly support mechanistic models that involve a tryptophan radical at this position (13,28,32). This tryptophan radical, generated close to the protein surface via LRET from a proposed Fe 4ϩ ϭO porphyrin -cation radical precursor, provides a powerful reactant, which is apparently capable of oxidizing also large molecular mass substrates, which, because of their large size, are not able to reach the active heme site of VP.…”

First Crystal Structure of a Fungal High-redox Potential Dye-decolorizing Peroxidase

“…enzyme before its first enzymatic turnover), it must have originated from an autocatalytic process during the first catalytic cycle. Trp-171 mutants (W171F, W171S) were shown to be completely inactive, confirming Trp-171 as the peripheral substrate oxidation site (44,45) for substrates like veratryl alcohol. In a similar way, a tyrosine on the enzyme surface acts as the substrate interaction site in lignin peroxidase from Trametes cervina (26).…”

“…An exposed oxidation site has been demonstrated extensively in a related heme enzyme viz. lignin peroxidase, where a hydroxylated tryptophan residue carries out the electron withdrawal from substrate molecules (43)(44)(45). In an electron density map of lignin peroxidase from Phanerochaete chrysosporium, strong electron density showed a hydroxylation at the C␤ position of the surfaceexposed Trp-171.…”

Reversible alkaline inactivation of lignin peroxidase involves the release of both the distal and proximal site calcium ions and bishistidine co-ordination of the haem

“…Some authors follow the nomenclature of Tien and Kirk [31] naming the isoenzymes H1, H2, H3, etc., based on their order of elution from a Pharmacia MonoQ ion-exchange column at pH 6.0. Other authors [18,32] refer to the isoenzyme's pI ; LiP H8, with a pI of 4.15, is known as LIP415. Others use their own numbering scheme ; Ritch et al [33], for example, refer to LiP H8 (LIP415) as LiP2.…”