Combustion” [7] and would involve peroxidases of the lignin peroxidase (LiP), manganese peroxidase (MnP) and versatile peroxidase (VP) families, collectively with other oxidoreductases [6, 8]. After some controversy in the past [9], by far the most current evidence on the involvement of peroxidases in lignin degradation comes from the availability of enormous sequencing tools applied to fungal genomes. The analysis of basidiomycete genomes shows the presence of the above ligninolytic peroxidase genes within the genomes of all common white-rot (ligninolytic) basidiomycetes sequenced to date, and their absence from all the brown-rot (cellulolytic) basidiomycete genomes [104]. Amongst the three peroxidase families LiP, first reported from Phanerochaete chrysosporium [15], and VP, described later from Pleurotus eryngii [16, 17], have attracted the highest interest because they’re able to degrade nonphenolic model compounds representing the main substructures in lignin (which include -O-4 alkyl-aryl Chromomycin A3 Purity & Documentation ethers) [180] by single-electron abstraction forming an aromatic cation radical [21], and subsequent C bond cleavage [22] (though MnP would act on the minor phenolic units). In the discovery of LiP, the substantial quantity of biochemical and molecular biology research on these enzymes typically applied easy aromatic substrates, including veratryl (3,4-dimethoxybenzyl) alcohol [235], and similar studies making use of the genuine lignin substrate are particularly rare [26]. A landmark in lignin biodegradation research was the identification of a solvent-exposed peroxidase residue, Trp171 in P. chrysosporium LiP (isoenzyme H8) [27, 28] and Trp164 in P. eryngii VP (isoenzyme VPL) [29], because the accountable for oxidative degradation of nonphenolic lignin model compounds by long-range electron transfer (LRET) in the protein Lesogaberan manufacturer surface to the heme cofactor from the H2O2-activated enzyme. This single-electron transfer generates a reactive tryptophanyl radical [30, 31], whose exposed nature would allow direct oxidationof the lignin polymer. Not too long ago, the authors have shown that removal of this aromatic residue lowers in distinctive extents the electron transfer from technical lignins (partially phenolic softwood and hardwood water-soluble lignosulfonates) to the peroxide-activated VP transient states (the so-called compounds I and II, CI and CII) [32, 33]. To clarify the part on the surface tryptophan residue in phenolicnonphenolic lignin degradation, stoppedflow reactions with the above VP as well as the corresponding tryptophan-less variant are performed within the present study applying native (underivatized) and permethylated acetylated (nonphenolic) softwood and hardwood lignosulfonates as enzyme substrates, together with lignosulfonate steady-state therapies analyzed by size-exclusion chromatography (SEC) and heteronuclear single quantum correlation (HSQC) two-dimensional nuclear magnetic resonance (2D-NMR).ResultsTransient kinetics of VP and its W164S variant: native ligninsPeroxidase catalytic cycle contains two-electron activation in the resting enzyme by H2O2 yielding CI, that is decreased back by way of CII with one-electron oxidation of two substrate molecules (Additional file 1: Figure S1a). These three enzyme forms present characteristic UV isible spectra (Added file 1: Figure S1b, c) that enable to calculate the kinetic constants for CI formation and CI CII reduction (see “Methods” section). The transient-state kinetic constants for the reaction of native lignosulfonates with H2O2-activated wild-type recombina.
