Structure and Enzymatic Characterization of CelD Cellulase from the Anaerobic Fungus Piromyces finnis


Anaerobic fungi found in the guts of large herbivores are prolific biomass degraders whose genomes harbor a wealth of carbohydrate-active enzymes (CAZymes), of which only a handful are structurally or biochemically characterized. Here, we report the structure and kinetic rate parameters for a glycoside hydrolase (GH) family 5 subfamily 4 enzyme (CelD) from Piromyces finnis, a modular, cellulosome-incorporated endoglucanase that possesses three GH5 domains followed by two C-terminal fungal dockerin domains (double dockerin). We present the crystal structures of an apo wild-type CelD GH5 catalytic domain and its inactive E154A mutant in complex with cellotriose at 2.5 and 1.8 Å resolution, respectively, finding the CelD GH5 catalytic domain adopts the (β/α)8-barrel fold common to many GH5 enzymes. Structural superimposition of the apo wild-type structure with the E154A mutant-cellotriose complex supports a catalytic mechanism in which the E154 carboxylate side chain acts as an acid/base and E278 acts as a complementary nucleophile. Further analysis of the cellotriose binding pocket highlights a binding groove lined with conserved aromatic amino acids that when docked with larger cellulose oligomers is capable of binding seven glucose units and accommodating branched glucan substrates. Activity analyses confirm P. finnis CelD can hydrolyze mixed linkage glucan and xyloglucan, as well as carboxymethylcellulose (CMC). Measured kinetic parameters show the P. finnis CelD GH5 catalytic domain has CMC endoglucanase activity comparable to other fungal endoglucanases with kcat = 6.0 ± 0.6 s−1 and Km = 7.6 ± 2.1 g/L CMC. Enzyme kinetics were unperturbed by the addition or removal of the native C-terminal dockerin domains as well as the addition of a non-native N-terminal dockerin, suggesting strict modularity among the domains of CelD.

ICB Affiliated Authors

Alexey Dementiev, Stephen P. Lillington, Shiyan Jin, Youngchang Kim, Robert Jedrzejczak, Karolina Michalska, Andrzej Joachimiak and Michelle A. O’Malley
Peer-Reviewed Article
Applied Microbiology and Biotechnology