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Towards an Understanding of Oxidative Damage in an α-L-Arabinofuranosidase of Trichoderma reesei: a Molecular Dynamics Approach

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Abstract

Trichoderma reesei is a “workhorse” fungus that produces glycosyl hydrolases (e.g., cellulases) at high titers for use in industrial bioprocessing. In this study, we focused on α-L-arabinofuranosidase, an enzyme important for the treatment of lignocellulosic biomass, but susceptible to oxidative damage that can occur during industrial processing. The molecular details that render this enzyme inactive have not yet been identified. To approach this issue, we used proteomics to identify amino acid residues that were oxidized after a relevant oxidative treatment (Fenton reaction). These oxidative modifications were included in the 3D protein structures, and using molecular dynamics simulations, we then studied the behaviors of non-modified and oxidized enzymes. These simulations showed significant alterations of the conformational stability of the protein when oxidized, as evidenced by changes in root mean square deviation (RMSD) and principal component analyses (PCA) trajectories. Likewise, enzyme-ligand interactions such as hydrogen bonds were greatly reduced in quantity and quality in the oxidized protein. Finally, free energy landscape plots showed that there was a more rugged energy surface in the oxidized protein, implying a less favorable reaction pathway. These results reveal the basis for loss of function in this carbohydrate active enzyme (CAZY) in the commercially relevant fungus T. reesei.

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Funding

The authors thank the Fulbright Commission and the Colombian Ministry of Science, Technology, and Innovation for the funding of the lead author. Research funding was from United States Department of Energy (US DOE) grant DE-SC0019427 and an Environmental Molecular Science Laboratory User Facility grant (EUP-50799), both sponsored by US DOE Biological and Environmental Research program.

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Authors and Affiliations

  1. Bioproducts and Biosystems Engineering, University of Minnesota, Saint Paul, MN, 55108, USA

    Jesus D. Castaño

  2. Marine and Coastal Research Institute, INVEMAR, Santa Marta, Colombia, 470006

    Jesus D. Castaño

  3. Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, WA, 99352, USA

    Mowei Zhou

  4. Plant and Microbial Biology, University of Minnesota, Saint Paul, MN, 55108, USA

    Jonathan Schilling

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  1. Jesus D. Castaño
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Contributions

Conceptualization, J.D.C.; methodology, J.D.C and M.Z.; writing—original draft preparation, J.D.C.; writing—review and editing, M.Z. and J.S.S.; project administration, J.S.S.; funding acquisition, J.S.S. All authors have read and agreed to the published version of the manuscript.

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Correspondence to Jonathan Schilling.

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Supplementary Information

Figure S1

Ligand-enzyme interactions for the non-modified and oxidized GH54 proteins determined with the Software Ligplot+. Amino acids that interact with the substrate in both proteins are shown in a gray box. (PNG 9840 kb)

High Resolution (TIFF 1379 kb)

Figure S2

Depiction of the atoms involved in the hydrogen bonds described in figure 5. The non-modified protein is shown on the left, while the oxidized protein is shown on the right. (PNG 108 kb)

High Resolution (TIF 1176 kb)

Table S1

(XLSX 12 kb)

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Castaño, J.D., Zhou, M. & Schilling, J. Towards an Understanding of Oxidative Damage in an α-L-Arabinofuranosidase of Trichoderma reesei: a Molecular Dynamics Approach. Appl Biochem Biotechnol 193, 3287–3300 (2021). https://doi.org/10.1007/s12010-021-03594-w

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