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Doctoral thesis2017Open access

GH7 cellobiohydrolases structural, functional and evolutional aspects

Borisova, Anna

Abstract

Fungal glycoside hydrolase family 7 cellobiohydrolases (GH7 CBH) are the workhorses of cellulose degradation and, thus, play a key role in the recycling of biomass on Earth. As central as these enzymes are to biomass degradation, they have become the cornerstone of modern industrial enzyme cocktails for biofuels processes. By combination of X-ray cystallography structure studies and other approaches, this thesis provides essential impact into understanding of sequence, structure and function correlation concepts in the GH7 family. Several new GH7 CBH structures were solved, ranging from the distant Amoebozoa to Trichoderma atroviride Cel7A, which is a close ortholog to the archetypal Trichoderma reesei Cel7A. Another ascomycete fungus, Scytalidium sp., exhibits new features of GH7 CBHs, never observed before. Namely, Scytalidium sp. Cel7A revealed O-glycosylation on the tunnel-enclosing B2 loop, and the loop can adopt different conformations, and even bend into the tunnel and obstruct cellulose binding. Trichoderma atroviride Cel7A ligand complex with thio-cellotriose represents a sliding intermediate during processive cellulose hydrolysis. Three new structures of GH7 CBHs labeled with a novel mechanism-based affinity tag confirm the proposed inactivation mechanism, presenting covalently bound enzyme-ligand complexes with Trichoderma reesei Cel7A and Scytalidium sp. Cel7A, and one complex with Trichoderma reesei Cel7A E217Q acid/base mutant where covalent bond is not formed. Two crystal structures of social amoeba GH7 CBHs appeared to be very similar to the well-studied fungal Trichoderma reesei Cel7A, despite the large evolutionary distance between these organisms. Phylogenetic analysis revealed high similarity between GH7 CBHs from different branches of the eukaryotic tree of life. Biochemical characterization and performance assays of the novel GH7 CBHs along with Molecular Dynamics (MD) simulations based on their structures, highlight important loop regions implicated in processivity, binding in the product sites, endo-/exo-initiation as well as thermostability and initiation of thermal unfolding. Reverse concervation analysis (RCA) identified potentially important evolutionary target sites in Trichoderma spp GH7 sequences. Loop dynamics and correlation with structure and function of GH7 CBH is thoroughly discussed in the thesis.

Keywords

cellulose, cellobiohydrolase, X-ray structure, molecular dynamics, Trichoderma, Dictyostelium, Scytalidium

Published in

Acta Universitatis Agriculturae Sueciae
2017, number: 2017:57ISBN: 978-91-7760-004-6, eISBN: 978-91-7760-005-3Publisher: Department of Molecular Sciences, Swedish University of Agricultural Sciences

    UKÄ Subject classification

    Biochemistry and Molecular Biology
    Structural Biology

    Permanent link to this page (URI)

    https://res.slu.se/id/publ/104103