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Binding and catalytic mechanisms of Veratryl alcohol oxidation by lignin peroxidase: A theoretical and experimental study

AuthorsRomero, Jefferson O.; Fernández-Fueyo, Elena CSIC ORCID; Ávila-Salas, Fabián; Recabarren, Rodrigo; Alzate-Morales, Jans; Martínez, Ángel T. CSIC ORCID
KeywordsPhanerochaete chrysosporium
Lignin peroxidase
Veratryl alcohol
Sited-directed mutagenesis
Interaction energy
Issue Date10-Jul-2019
CitationComputational and Structural Biotechnology 17:1066-1074 (2019)
AbstractLignin peroxidase (LiP) and its natural substrate veratryl alcohol (VA) play a crucial role in lignin degradation by white-rot fungi. Understanding the molecular determinants for the interaction of this enzymewith its substrates is essential in the rational design of engineered peroxidases for biotechnological application. Here, we combine computational and experimental approaches to analyze the interaction of Phanerochaete chrysosporium LiP (isoenzyme H8)with VA and its radical cation (VA•+, resulting fromsubstrate oxidation by the enzyme). Interaction energy calculations at semiempirical quantum mechanical level (SQM) between LiP and VA/VA•+ enabled to identify those residues at the acidic environment of catalytic Trp171 involved in the main interactions. Then, a battery of variants, with single and multiple mutations at these residues (Glu168, Asp165, Glu250, Asp264,and Phe267), was generated by directed mutagenesis, and their kinetics parameters were estimated on VA and two additional substrates. The experimental results show that Glu168 and Glu250 are crucial for the binding of VA, with Glu250 also contributing to the turnover of the enzyme. The experimental resultswere further rationalized through new calculations of interaction energies between VA/VA•+ and LiP with each of the single mutations.Finally, the delocalization of spin density was determined with quantum mechanics/molecular mechanics calculations (QM/MM), further supporting the contribution of Glu250 to VA oxidation at Trp171.
Description9 p.-3 fig.-4 tab.
Publisher version (URL)https://doi.org/10.1016/j.csbj.2019.07.002
Appears in Collections:(CIB) Artículos
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