Structure-function studies of GH7 cellulases, key enzymes in the global carbon cycle

Abstract

Enzyme mixtures used for lignocellulosic ethanol production are most commonly derived from filamentous fungi, and enzymes from the glycoside hydrolase family 7 (GH7) constitute the most abundant components in these cocktails. In this thesis I have aimed to increase our understanding of this enzyme family, with focus on the interrelation between their structure and function. In a study of the two model enzymes Trichoderma reesei Cel7A (TreCel7A) and Phanerochaete chrysosporium Cel7D (Pch7D), we determined factors governing the idiosyncratic behavior of these enzymes on commonly used model compounds, and by using fluorescence titration, enzyme kinetics, structure determination and molecular dynamics simulations found specific structural features connected to nonproductive binding, playing a major role in enzyme activity on these compounds. We also determined the molecular structure of a GH7 enzyme RsSymEG1, belonging to a group of smaller GH7 endoglucanases with previously unknown structure architecture, and originating from symbiotic protozoa of wood eating lower termites. The X-ray crystal structure revealed a configuration with several key differences to previously known GH7 structures, and will aid in modelling and engineering of enzymes in this so far little-known group of enzymes. A further look into this group of sequences, as well as other GH7 enzymes found in the termite symbiont protists, also revealed previously unknown details about the evolution of this ancient enzyme family. Furthermore, we explored single molecule imaging of the model enzyme TreCel7A with novel imaging methods, providing a first proof-of-concept of using fluorescence resonance energy transfer (FRET) for the study of inter-domain dynamics of this enzyme, as well as total internal reflection dark-field microscopy (TIRDFM) for imaging enzyme movement on cellulose surface at ultra-high temporal resolutions.

The crystal structure of RsSymEG1 reveals a unique form of smaller GH7 endoglucanases alongside GH7 cellobiohydrolases in protist symbionts of termites

“…The figure was created using ESPript [68], with further loop and residue annotations added in Microsoft PowerPoint. Figure and legend reproduced from [70] with modifications. The copyrights to the doctoral thesis [70] and the original image this has been reproduced from are held by the author, Topi Haataja.…”

“…Figure and legend reproduced from [70] with modifications. The copyrights to the doctoral thesis [70] and the original image this has been reproduced from are held by the author, Topi Haataja. The authors and The FEBS Journal have been granted permission to reproduce the image.…”

“…The tree was built with IQ-TREE web server using the default settings (bootstrap alignments n = 1000), based on an alignment generated with MAFFT-L-INS-I algorithm on the MAFFT web server, and visualized using iTOL web server[64,66,67]. Figure and legend reproduced from[70] with modifications. The copyrights to the doctoral thesis[70] and the original image this has been reproduced from are held by the author, Topi Haataja.…”