Biochemical and Structural Characterizations of Two Dictyostelium Cellobiohydrolases from the Amoebozoa Kingdom Reveal a High Level of Conservation between Distant Phylogenetic Trees of Life

Abstract

Glycoside hydrolase family 7 (GH7) cellobiohydrolases (CBHs) are enzymes commonly employed in plant cell wall degradation across eukaryotic kingdoms of life, as they provide significant hydrolytic potential in cellulose turnover. To date, many fungal GH7 CBHs have been examined, yet many questions regarding structure-activity relationships in these important natural and commercial enzymes remain. Here, we present the crystal structures and a biochemical analysis of two GH7 CBHs from social amoeba: Dictyostelium discoideum Cel7A (DdiCel7A) and Dictyostelium purpureum Cel7A (DpuCel7A). DdiCel7A and DpuCel7A natively consist of a catalytic domain and do not exhibit a carbohydrate-binding module (CBM). The structures of DdiCel7A and DpuCel7A, resolved to 2.1 Å and 2.7 Å, respectively, are homologous to those of other GH7 CBHs with an enclosed active-site tunnel. Two primary differences between the Dictyostelium CBHs and the archetypal model GH7 CBH, Trichoderma reesei Cel7A (TreCel7A), occur near the hydrolytic active site and the product-binding sites. To compare the activities of these enzymes with the activity of TreCel7A, the family 1 TreCel7A CBM and linker were added to the C terminus of each of the Dictyostelium enzymes, creating DdiCel7A CBM and DpuCel7A CBM , which were recombinantly expressed in T. reesei. DdiCel7A CBM and DpuCel7A CBM hydrolyzed Avicel, pretreated corn stover, and phosphoric acid-swollen cellulose as efficiently as TreCel7A when hydrolysis was compared at their temperature optima. The K i of cellobiose was significantly higher for DdiCel7A CBM and DpuCel7A CBM than for TreCel7A: 205, 130, and 29 M, respectively. Taken together, the present study highlights the remarkable degree of conservation of the activity of these key natural and industrial enzymes across quite distant phylogenetic trees of life. IMPORTANCE GH7CBHs are among the most important cellulolytic enzymes both in nature and for emerging industrial applications for cellulose breakdown. Understanding the diversity of these key industrial enzymes is critical to engineering them for higher levels of activity and greater stability. The present work demonstrates that two GH7 CBHs from social amoeba are surprisingly quite similar in structure and activity to the canonical GH7 CBH from the model biomass-degrading fungus T. reesei when tested under equivalent conditions (with added CBM-linker domains) on an industrially relevant substrate. Dictyostelia are a class of social amoebae (historically known as slime molds) containing four different groups of terrestrial bacterivores from the kingdom Amoebozoa. During vegetative growth, dictyostelia exist as single-celled organisms; upon starvation, a lack of nutrients becomes preventive for vegetative growth and the cells aggregate into a multicellular slug. Slugs have a defined posterior and anterior, have the ability to migrate, are sensitive to light and temperature, and exhibit an innate immune system. When conditions are sufficiently severe, the slug can form a fruiting body, w...

Improving the thermal stability of cellobiohydrolase Cel7A from Hypocrea jecorina by directed evolution

“…None of the mutations in the FCA398 catalytic domain are at sites that are completely conserved in the phylogenetically diverse GH7 CBH sequences, examined in the recent paper by Hobdey et al (52). Only one residue, Asn 49 , is conserved among the known GH7 CBH structures.…”

Systematic deletions in the cellobiohydrolase (CBH) Cel7A from the fungus Trichoderma reesei reveal flexible loops critical for CBH activity

“…1A. Several earlier studies have suggested that the functional differences between CBHs and EGs can be attributed to loop configuration (25). As an example, Meinke et al (26) explored the importance of the loops for distinguishing between exo-and endolytic activity in a GH6 cellulase.…”

pH profiles of cellulases depend on the substrate and architecture of the binding region

“…The results in Figure show that higher loads of both substrates promote activity in the acidic range. In other words, Cel7A was generally more tolerant to low pH, when the substrate was plentiful and most enzymes were engaged in a complex ( K M for Cel7A acting on pNPL is around 2 mM (Hobdey et al, ; Sørensen et al, ). Figure also underscores that binding of a cellulose strand that fills the entire substrate‐binding tunnel brings about higher tolerance to low pH compared with the binding of a small substrate analog, which only covers a small part of the tunnel (Jalak & Väljamäe, ).…”

“…This could, at least in part, be because most of these pH profiles are based on measurements at a single load of cellulose, although this is known to limit interpretations of pH-activity relationship (Knowles & Jencks, 1976). We conclude that there is no clear picture of whether pH profiles of cellulases depends on the nature of the substrate, and in light of the distinctive effect of the substrate on kinetic parameters (Becker et al, 2001;Borisova et al, 2018;Hobdey et al, 2016;Sørensen et al, 2015), systematic work on pH effects appears warranted. To address this, we measured pH profiles for selected cellulases over different ranges of temperature and substrate loads for both soluble and insoluble substrates.…”

“…Perhaps for that reason, cellulases are often characterized on soluble substrate analogs that are convenient to work with and provide results, which can be readily rationalized by conventional enzyme kinetic theory. The use of soluble substrate has been particularly dominant in pH studies, and many works have reported traditional bell-shaped pH profiles with an apex (i.e., optimal pH value) around pH 4-5 (see e.g., Becker et al, 2001;Boer & Koivula, 2003;Borisova et al, 2018;Hobdey et al, 2016;Momeni et al, 2013;Schülein, 1997). Work with soluble substrate has also identified cellulase wild-types with higher (Cockburn, Vandenende, & Clarke, 2010) or lower (Pellegrini et al, 2015;Texier, Dumon, Neugnot-Roux, Maestracci, & O'Donohue, 2012) pH optima, and engineered enzyme variants with upwards shifts of the optimal value (Becker et al, 2001;Cockburn et al, 2010;Qin, Wei, Song, & Qu, 2008).…”