Abstract
BackgroundLignin peroxidases catalyze a variety of reactions, resulting in cleavage of both β-O-4′ 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-O-4′ 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.ResultsUsing a nanostructure initiator mass spectrometry assay that provides quantification of bond breaking in a phenolic model lignin dimer we found that catalysis of degradation of the dimer to products by an acid-stabilized variant of lignin peroxidase isozyme H8 increased 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% β-O-4′ ether bond cleavage, 27.0% Cα-C1 carbon bond cleavage, and 3.6% Cα-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 protonation of aliphatic hydroxyl groups under which extremely acidic conditions resulted in lower energetic barriers for bond-cleavages, particularly β-O-4′ bonds.ConclusionThese coupled experimental results and theoretical explanations suggest pH is a key driving force for selective and efficient lignin peroxidase isozyme H8 catalyzed depolymerization of the phenolic lignin dimer and further suggest that engineering of lignin peroxidase isozyme H8 and other enzymes involved in lignin depolymerization should include targeting stability at low pH.
Highlights
Lignin peroxidases catalyze a variety of reactions, resulting in cleavage of both β-O-4′ 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
We report on our use of quantum calculations and ab initio molecular dynamic (AIMD) simulations to generate a fundamental understanding of how pH assist bond cleavage and show the roles of low pH are to i) provide a thermodynamically favorable condition for the formation of a cation radical intermediate required for energy-favorable degradation of lignin, ii) pH-assisted formation of protonated cationic radical intermediate at both phenolic and aliphatic hydroxyl groups, resulting in higher cleavage frequencies for various bond types in lignin, especially β-O-4′ ether bonds
Results and discussion pH‐controlled catalysis of bond cleavage by the lignin peroxidase from P. chrysosporium Lignin is an unusual biopolymer because of its structural heterogeneity, which has limited detailed studies of lignin-degrading enzymes in terms of their ability to catalyze the breaking of specific bond types
Summary
Lignin peroxidases catalyze a variety of reactions, resulting in cleavage of both β-O-4′ 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. We report on combined experimental and theoretical studies of the effect of pH on the enzyme-catalyzed cleavage of β-O-4′ 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. Some microorganisms involved in biomass degradation actively modify the pH of their environment via secretion of acids and bases, and fungi produce organic acids, resulting in significant acidification of conditions in their microenvironment, which is thought to play many key roles in nature [7,8,9]. Two Basidiomycota known to degrade lignin, Phanerochaete chrysosporium, and Trametes menziesii, have been shown to acidify their environment to pH 2 or lower, implicating the pH of the fungal environment as a key driving force for the biological breakdown of biomass and for depolymerization of lignin catalyzed by LiP, MnP, VP and laccases [10]
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