In:
Applied and Environmental Microbiology, American Society for Microbiology, Vol. 82, No. 11 ( 2016-06), p. 3395-3409
Kurzfassung:
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 ( Ddi Cel7A) and Dictyostelium purpureum Cel7A ( Dpu Cel7A). Ddi Cel7A and Dpu Cel7A natively consist of a catalytic domain and do not exhibit a carbohydrate-binding module (CBM). The structures of Ddi Cel7A and Dpu Cel7A, 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 ( Tre Cel7A), occur near the hydrolytic active site and the product-binding sites. To compare the activities of these enzymes with the activity of Tre Cel7A, the family 1 Tre Cel7A CBM and linker were added to the C terminus of each of the Dictyostelium enzymes, creating Ddi Cel7A CBM and Dpu Cel7A CBM , which were recombinantly expressed in T. reesei . Ddi Cel7A CBM and Dpu Cel7A CBM hydrolyzed Avicel, pretreated corn stover, and phosphoric acid-swollen cellulose as efficiently as Tre Cel7A when hydrolysis was compared at their temperature optima. The K i of cellobiose was significantly higher for Ddi Cel7A CBM and Dpu Cel7A CBM than for Tre Cel7A: 205, 130, and 29 μM, 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 GH7 CBHs 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.
Materialart:
Online-Ressource
ISSN:
0099-2240
,
1098-5336
DOI:
10.1128/AEM.00163-16
Sprache:
Englisch
Verlag:
American Society for Microbiology
Publikationsdatum:
2016
ZDB Id:
223011-2
ZDB Id:
1478346-0
SSG:
12
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