Background: The ascomycete fungus Trichoderma reesei is the predominant source of enzymes for industrial conversion of lignocellulose. Its glycoside hydrolase family 7 cellobiohydrolase (GH7 CBH) TreCel7A constitutes nearly half of the enzyme cocktail by weight and is the major workhorse in the cellulose hydrolysis process. The orthologs from Trichoderma atroviride (TatCel7A) and Trichoderma harzianum (ThaCel7A) show high sequence identity with TreCel7A, ~ 80%, and represent naturally evolved combinations of cellulose-binding tunnel-enclosing loop motifs, which have been suggested to influence intrinsic cellobiohydrolase properties, such as endo-initiation, processivity, and off-rate.

Results: The TatCel7A, ThaCel7A, and TreCel7A enzymes were characterized for comparison of function. The catalytic domain of TatCel7A was crystallized, and two structures were determined: without ligand and with thio-cellotriose in the active site. Initial hydrolysis of bacterial cellulose was faster with TatCel7A than either ThaCel7A or TreCel7A. In synergistic saccharification of pretreated corn stover, both TatCel7A and ThaCel7A were more efficient than TreCel7A, although TatCel7A was more sensitive to thermal inactivation. Structural analyses and molecular dynamics (MD) simulations were performed to elucidate important structure/function correlations. Moreover, reverse conservation analysis (RCA) of sequence diversity revealed divergent regions of interest located outside the cellulose-binding tunnel of Trichoderma spp. GH7 CBHs.

Conclusions: We hypothesize that the combination of loop motifs is the main determinant for the observed differences in Cel7A activity on cellulosic substrates. Fine-tuning of the loop flexibility appears to be an important evolutionary target in Trichoderma spp., a conclusion supported by the RCA data. Our results indicate that, for industrial use, it would be beneficial to combine loop motifs from TatCel7A with the thermostability features of TreCel7A. Furthermore, one region implicated in thermal unfolding is suggested as a primary target for protein engineering.

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Published in Biotechnology for Biofuels, v. 11, 5, p. 1-22.

© The Author(s) 2018

This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

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This material is based upon work supported by the Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning (Formas; Grant Number 213-2013-1607; PI: JS); Russian Science Foundation (Grant Number 16-14-00109; PI: AAK); the National Science Foundation (NSF) under Grant Number 1552355 (former PI: CMP); Novo Nordisk Foundation (Grant Number NNF15OC0016606; PI: PW) and Innovation Fund Denmark (Grant Number 5150-00020B; PI: PW). This material is also based upon work supported by (while CMP is serving at) the NSF. Any opinion, findings, and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the NSF. Computing resources for MD simulations were provided by the University of Kentucky and NSF Extreme Science and Engineering Discovery Environment (XSEDE), which is supported by NSF Grant Number ACI-1548562.

Related Content

The protein structure models and structure factors are available in the Protein Data Bank (http://wwpdb.org/) under accession codes 5O5D and 5O59. All other data generated and/or analyzed during the current study are either included in this published article and its Additional files, or available in public databases (e.g. Embank, Uniprot, JGI MycoCosm portal), or available from the corresponding author on reasonable request.

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