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. 2019 Jul 10:17:1066-1074.
doi: 10.1016/j.csbj.2019.07.002. eCollection 2019.

Binding and Catalytic Mechanisms of Veratryl Alcohol Oxidation by Lignin Peroxidase: A Theoretical and Experimental Study

Jefferson O Romero  1   2 , Elena Fernández-Fueyo  3   4 , Fabián Avila-Salas  5   6 , Rodrigo Recabarren  1 , Jans Alzate-Morales  1 , Angel T Martínez  3
Affiliations

Binding and Catalytic Mechanisms of Veratryl Alcohol Oxidation by Lignin Peroxidase: A Theoretical and Experimental Study

Jefferson O Romero et al. Comput Struct Biotechnol J. .

Abstract

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.

Keywords: Interaction energy; Lignin peroxidase; Phanerochaete chrysosporium; QM/MM; SQM; Sited-directed mutagenesis; Veratryl alcohol.

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Figures

Unlabelled Image
Graphical abstract
Fig. 1
Fig. 1
Residues located at <5.5 Å from Trp171 in wild-type LiPH8 (a), and its in silico mutated systems (b-f).
Fig. 2
Fig. 2
Main π-π stacking intermolecular interactions in the LiPH8 Trp171-VA•+ complexes with the best interaction energies, corresponding to: (a,b) wild-type LiP region, (c) E250Q variant region, and (d, e) D264N variant region.
Fig. 3
Fig. 3
QM/MM electron spin distribution on LiPH8 Trp171 and heme cofactor. (a) Boxplot representation of Trp171 spin density in wild-type LiPH8 and its E250Q variant. (b) Spin density for the three unpaired electrons in LiPH8 (c) Spin density for the three unpaired electron in E250Q.
See this image and copyright information in PMC

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