Abstract
Background: Lignin peroxidases catalyze a variety of reactions, including cleavage of both β-aryl ether bonds and C–C bonds in lignin, both of which are essential for depolymerizing lignin into fragments amendable to biological or chemical upgrading to valuable products. Studies of the specificity of lignin peroxidases to catalyze these various reactions and the role reaction conditions such as pH play have been limited by the lack of assays that allow quantification of specific bond-breaking events. The subsequent theoretical understanding of the underlying mechanisms by which pH modulates the activity of lignin peroxidases remains nascent. Here, we report on combined experimental and theoretical studies of the effect of pH on the enzyme-catalyzed cleavage of β-aryl ether bonds and of C–C bonds by a lignin peroxidase isozyme H8 from Phanerochaete chrysosporium and an acid stabilized variant of the same enzyme. Results: Using a nanostructure initiator mass spectrometry assay that provides quantification of bond cleavages in a phenolic model lignin dimer we found that degradation of the lignin dimer was greatly enhanced at lower pH, and the acid-stable lignin peroxidase isozyme H8 increased the degradation of the lignin dimer to products from 38.4 % at pH 5 to 92.5% at pH 2.6. At pH 2.6, the observed product distribution resulted from 65.5% b -aryl ether bond cleavage, 27.0% C a -aryl carbon bond cleavage and 3.6% C a -oxidation as by-product . Using ab initio molecular dynamic simulations and climbing-image Nudge Elastic Band based transition state searches, we suggest the effect of lower pH is via hydration of hydronium cation on aliphatic and phenolic hydroxyl groups under, which extremely acidic conditions resulted in lower energetic barriers for bond-cleavages, especially β-ether bonds. Conclusion: These coupled experimental results and theoretical explanations suggest pH is a key driving force for selective and efficient lignin peroxidase isozyme H8 catalyzed depolymerization of lignin and further suggest that lignin peroxidase isozyme H8 engineering efforts include targeting stability at low pH.
Molecular cloning and sequences of lignin peroxidase genes of Phanerochaete chrysosporium.
