TY - JOUR
T1 - Binding and Catalytic Mechanisms of Veratryl Alcohol Oxidation by Lignin Peroxidase
T2 - A Theoretical and Experimental Study
AU - Romero, Jefferson O.
AU - Fernández-Fueyo, Elena
AU - Avila-Salas, Fabián
AU - Recabarren, Rodrigo
AU - Alzate-Morales, Jans
AU - Martínez, Angel T.
PY - 2019
Y1 - 2019
N2 - Lignin 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 enzyme with 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 from substrate 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 results were 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.
AB - Lignin 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 enzyme with 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 from substrate 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 results were 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.
KW - Interaction energy
KW - Lignin peroxidase
KW - Phanerochaete chrysosporium
KW - QM/MM
KW - Sited-directed mutagenesis
KW - SQM
KW - Veratryl alcohol
UR - http://www.scopus.com/inward/record.url?scp=85070393110&partnerID=8YFLogxK
U2 - 10.1016/j.csbj.2019.07.002
DO - 10.1016/j.csbj.2019.07.002
M3 - Article
AN - SCOPUS:85070393110
SN - 2001-0370
VL - 17
SP - 1066
EP - 1074
JO - Computational and Structural Biotechnology Journal
JF - Computational and Structural Biotechnology Journal
ER -