| 1. |
- Haataja, Topi, et al.
(författare)
-
Enzyme kinetics by GH7 cellobiohydrolases on chromogenic substrates is dictated by non-productive binding : insights from crystal structures and MD simulation
- 2023
-
Ingår i: The FEBS Journal. - : John Wiley & Sons. - 1742-464X .- 1742-4658. ; 290:2, s. 379-399
-
Tidskriftsartikel (refereegranskat)abstract
- Cellobiohydrolases (CBHs) in the glycoside hydrolase family 7 (GH7) (EC3.2.1.176) are the major cellulose degrading enzymes both in industrial settings and in the context of carbon cycling in nature. Small carbohydrate conjugates such as p-nitrophenyl-beta-d-cellobioside (pNPC), p-nitrophenyl-beta-d-lactoside (pNPL) and methylumbelliferyl-beta-d-cellobioside have commonly been used in colorimetric and fluorometric assays for analysing activity of these enzymes. Despite the similar nature of these compounds the kinetics of their enzymatic hydrolysis vary greatly between the different compounds as well as among different enzymes within the GH7 family. Through enzyme kinetics, crystallographic structure determination, molecular dynamics simulations, and fluorometric binding studies using the closely related compound o-nitrophenyl-beta-d-cellobioside (oNPC), in this work we examine the different hydrolysis characteristics of these compounds on two model enzymes of this class, TrCel7A from Trichoderma reesei and PcCel7D from Phanerochaete chrysosporium. Protein crystal structures of the E212Q mutant of TrCel7A with pNPC and pNPL, and the wildtype TrCel7A with oNPC, reveal that non-productive binding at the product site is the dominating binding mode for these compounds. Enzyme kinetics results suggest the strength of non-productive binding is a key determinant for the activity characteristics on these substrates, with PcCel7D consistently showing higher turnover rates (k(cat)) than TrCel7A, but higher Michaelis-Menten (K-M) constants as well. Furthermore, oNPC turned out to be useful as an active-site probe for fluorometric determination of the dissociation constant for cellobiose on TrCel7A but could not be utilized for the same purpose on PcCel7D, likely due to strong binding to an unknown site outside the active site.
|
|
| 2. |
- Haataja, Topi
(författare)
-
Structure-function studies of GH7 cellulases, key enzymes in the global carbon cycle
- 2023
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)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 keydifferences 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 ofthis ancient enzyme family.Furthermore, we explored single molecule imaging of the model enzymeTreCel7A with novel imaging methods, providing a first proof-of-concept of usingfluorescence resonance energy transfer (FRET) for the study of inter-domaindynamics of this enzyme, as well as total internal reflection dark-field microscopy(TIRDFM) for imaging enzyme movement on cellulose surface at ultra-hightemporal resolutions.
|
|
| 3. |
- Haataja, Topi, et al.
(författare)
-
The crystal structure of RsSymEG1 reveals a unique form of smaller GH7 endoglucanases alongside GH7 cellobiohydrolases in protist symbionts of termites
- 2024
-
Ingår i: The Febs Journal. - 1742-464X .- 1742-4658. ; 291, s. 1168-1185
-
Tidskriftsartikel (refereegranskat)abstract
- Glycoside hydrolase family 7 (GH7) cellulases are key enzymes responsible for carbon cycling on earth through their role in cellulose degradation and constitute highly important industrial enzymes as well. Although these enzymes are found in a wide variety of evolutionarily distant organisms across eukaryotes, they exhibit remarkably conserved features within two groups: exo-acting cellobiohydrolases and endoglucanases. However, recently reports have emerged of a separate clade of GH7 endoglucanases from protist symbionts of termites that are 60-80 amino acids shorter. In this work, we describe the first crystal structure of a short GH7 endoglucanase, RsSymEG1, from a symbiont of the lower termite Reticulitermes speratus. A more open flat surface and shorter loops around the non-reducing end of the cellulose-binding cleft indicate enhanced access to cellulose chains on the surface of cellulose microfibrils. Additionally, when comparing activities on polysaccharides to a typical fungal GH7 endoglucanase (Trichoderma longibrachiatum Cel7B), RsSymEG1 showed significantly faster initial hydrolytic activity. We also examine the prevalence and diversity of GH7 enzymes that the symbionts provide to the termite host, compare overall structures and substrate binding between cellobiohydrolase and long and short endoglucanase, and highlight the presence of similar short GH7s in other organisms.
|
|
