Two Substrate Interaction Sites in Lignin Peroxidase Revealed by Site-Directed Mutagenesis

Abstract

It has been shown recently that Trp171 of lignin peroxidase (LiP) is hydroxylated at the Cbeta position [Blodig, W., Doyle, W. A., Smith, A. T., Winterhalter, K., Choinowski, T., and Piontek, K. (1998) Biochemistry 37, 8832-8838]. Comparative experiments, carried out on both wild-type fungal and recombinant LiP isoenzyme H8 (LiPH8), indicate that the process of hydroxylation is autocatalytic and that Trp171 may be implicated in catalysis. The role of this residue has therefore been examined using site-directed mutagenesis to obtain recombinant enzymes with Trp171 substituted by Phe or Ser (W171F and W171S LiPH8, respectively). The wild-type recombinant enzyme (LiPH8) was analyzed in solution using 1H NMR spectroscopy and its integrity confirmed prior to the kinetic and spectroscopic characterization of LiPH8 mutants. A charge neutralization mutation in the "classical heme edge" substrate access channel of LiP, in which Glu146 was substituted by Gly (E146G LiPH8), showed substantial activity with respect to veratryl alcohol (VA) oxidation and a marked (2.4 pH units) increase in pKa for the oxidation of a negatively charged difluoroazo dye. More surprisingly, the Trp171 LiPH8 mutants W171F and W171S LiPH8 were found to have lost all activity with VA as substrate, and compounds I and II were unable to react with VA. Both mutants, however, retained substantial activity with two dye substrates. These data provide the first direct evidence for the existence of two distinct substrate interaction sites in LiP, a heme-edge site typical of those encountered in other peroxidases and a second, novel site centered around Trp171 which is required for the oxidation of VA. Stopped-flow kinetic studies showed that all the mutants examined reacted normally with hydrogen peroxide to give a porphyrin cation radical (compound I). However, the rapid phase of spontaneous compound I reduction (2.3 s-1), typical of wild-type LiP, was absent in the Trp171 mutants, strongly suggesting that an electron-transfer pathway must exist within the protein leading from the heme to a surface site in close proximity to Trp171. The kinetic competence of such a pathway is dependent on interaction of the enzyme with VA, at or near Trp171.

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

“…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.…”

Progress and obstacles in the production and application of recombinant lignin-degrading peroxidases

“…22 However, the extensive screening required to identify mutant production strains, as well as contamination with the native enzyme, led to disappointing results. The model bacterium Escherichia coli has been extensively developed as an expression platform for fungal enzymes, and many recombinant enzymes have been isolated successfully from inclusion bodies and refolded with 1-28% efficiency [23][24][25][26][27][28][29][30] and maximum yields of 1.5-14 mg L ¡1 after in vitro activation and purification. 29 A soluble form of a VP was produced using a thioredoxin tag 31,32 or following intensive screening.…”

“…However, the degradation of these simple aromatic compounds may require different catalytic mechanisms, which makes it difficult to extrapolate the results to typical lignin and lignocellulose samples. For example, LiP has two substrate interaction sites: a heme-edge site which is typical for peroxidases and a second site for the oxidation of VA. 24 The optimization of enzyme activity by screening will only select for increases in activity against the test substrate under the specific conditions of the test and will not necessarily have a positive impact on the degradation of typical lignin substrates found in plant biomass or organosolv lignin. It is also clear that the successful degradation of lignin and lignocellulose requires multiple peroxidases and other lignin-degrading enzymes and accessory proteins.…”

Microbial degradation of lignin: Role of lignin peroxidase, manganese peroxidase, and laccase

“…Doyle and his group 19) recently found that , degrades the nonphenolic lignin model veratrylglycerol-β -guaiacyl ether yielding veratryl aldehyde, and oxidizes veratryl alcohol and p-dimethoxybenzene to veratryl aldehyde and p-benzoquinone respectively as LiP does. A review on lignin conversion by MnP has been published recently.…”

“…Doyle and his group 19) recently found that by cultivating wood-rotting fungi in an agar medium containing several phenolic compounds, such as gallic acid, tannic acid, and hydroquinone, that white-rot fungi produced a large darkened zone around the mycerial mat, but no zone of darkening was associated with the growth of brown-rot fungi. Davidson et al 37) subsequently investigated the reaction using 210 species of wood-rotting fungi, and concluded that the white-rotting type coincides with Bavendamm's reaction in general, and that the reaction is helpful in identifying fungi.…”