Experimental and theoretical insights into the effects of pH on catalysis of bond-cleavage by the lignin peroxidase isozyme H8 from Phanerochaete chrysosporium

Background 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. 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. Results Using 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. 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 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.

Experimental and theoretical insights into the effects of pH on catalysis of bond-cleavage by the lignin peroxidase isozyme H8 from Phanerochaete chrysosporium

Background: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. 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.Results: Using 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% b-O-4’ ether bond cleavage, 27.0% Ca-C1 carbon bond cleavage and 3.6% Ca-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. 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 the phenolic lignin dimer and further suggest that engineering of lignin peroxidase isozyme H8 and other enzymes involved lignin depolymerization should include targeting stability at low pH.

Experimental and theoretical insights into the effects of pH on catalysis of bond-cleavage by the lignin peroxidase isozyme H8 from Phanerochaete chrysosporium

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.

Effects of jinggangmycin on peroxidase and esterase isozymes and phytotoxin from Rhizoctonia solani AG-1 IA, the causal agent of rice sheath blight. Pirinç kabuğu yanığı nedeni Rhizoctonia solani AG-1 IA tarafından üretilen peroksidaz, esteraz izozimleri ve fitotoksinler üzerinde jinggangmycin etkisi

Objective: To explore the effects of jinggangmycin on the pathogenicity of rice sheath blight.Methods: The effects of jinggangmycin on peroxidase and esterase isozymes from Rhizoctonia solani Kuhn AG-1 IA were compared using non-denaturing polyacrylamide gel electrophoresis (PAGE). Rs-toxins were obtained from the improved Richard medium amended with jinggangmycin at different concentrations. Subsequently, bioassays of Rs-toxin, including virulence to rice tissues, seedling wilting and inhibition to the seed germination were conducted.Results: The results of PAGE showed that when the jinggangmycin concentration increased to 50 μg/mL, two of peroxidase isozyme bands became apparently weaker compared with the controls, indicating that jinggangmycin could considerably weaken the activity of the peroxidase isozyme. In addition, three extra bands of esterase isozyme were induced by jinggangmycin when the concentration increased to 50 μg/mL, indicating that jinggangmycin could affect the metabolism of esterase isozyme. The bioassays of Rs-toxin showed that jinggangmycin could weaken the virulence of Rs-toxin in rice tissues.Conclusion: It was concluded that the control role of jinggangmycin in rice sheath blight might be due to the effects of jinggangmycin on the peroxidase and esterase isozymes and Rs-toxin virulence.