The substrate binding sites of lignin peroxidase and manganese peroxidase.

摘要

The lignin-degrading fungus, Phanerochaete chrysosporium, secretes two families of extracellular peroxidases, lignin peroxidase (LiP) and manganese peroxidase (MnP), which are major enzymatic components of its extracellular lignin degradation system.; The role of amino acid Arg177 in the binding and oxidation of Mn II by MnP was investigated by constructing several site-directed mutant enzymes. These variant proteins were analyzed by UV-vis and resonance Raman spectroscopy, and steady state and transient-state kinetics. Altered kinetic characteristics strongly suggest that Arg177 is important in MnII oxidation, orienting the MnII ligand Glu35 for efficient Mn II binding.; A homologous expression system was developed for UP in order to study structure-function relationships in this enzyme via site-directed mutagenesis. The expression vector contained the Schizophyllum commune ura1 gene as a selectable marker and the coding regions of the lipH8 or lipH2 genes, which were placed under the control of the glyceraldehyde-3-phosphate dehydrogenase gene (gpd) promoter. Transformants of P. chrysosporium were selected for the production of extracellular, active recombinant LiP enzymes. Spectral and kinetic properties of the recombinant enzymes are essentially identical to the respective LiPH8 and LiPH2 wild-type enzymes.; Recombinant LiPH2 was used, together with wild-type LiPH2 and LiPH8, to investigate claims that LiP is able to oxidize MnII. Spectral and kinetic analysis of purified enzyme preparations reveal that LiP is unable to use MnII as a productive substrate.; Mutant LiPH8 enzymes were produced to investigate the site for veratryl alcohol (VA) oxidation in LiP. Analysis of the site-directed mutations, H82A and Q222A, suggests that the heme access channel is an unlikely VA binding site in LiP. Analysis of the W171A and F267L mutant enzymes demonstrated that Trp171 is essential for VA oxidation and indicated that F267 affects VA binding, confirming that the Trp171 site is the productive site for VA oxidation.