CelI, a Noncellulosomal Family 9 Enzyme from Clostridium thermocellum , Is a Processive Endoglucanase That Degrades Crystalline Cellulose

Abstract

The family 9 cellulase gene celI of Clostridium thermocellum, was previously cloned, expressed, and characterized (G. P. Hazlewood, K. Davidson, J. I. Laurie, N. S. Huskisson, and H. J. Gilbert, J. Gen. Microbiol. 139:307-316, 1993). We have recloned and sequenced the entire celI gene and found that the published sequence contained a 53-bp deletion that generated a frameshift mutation, resulting in a truncated and modified C-terminal segment of the protein. The enzymatic properties of the wild-type protein were characterized and found to conform to those of other family 9 glycoside hydrolases with a so-called theme B architecture, where the catalytic module is fused to a family 3c carbohydrate-binding module (CBM3c); CelI also contains a C-terminal CBM3b. The intact recombinant CelI exhibited high levels of activity on all cellulosic substrates tested, with pH and temperature optima of 5.5 and 70°C, respectively, using carboxymethylcellulose as a substrate. Native CelI was capable of solubilizing filter paper, and the distribution of reducing sugar between the soluble and insoluble fractions suggests that the enzyme acts as a processive cellulase. A truncated form of the enzyme, lacking the C terminal CBM3b, failed to bind to crystalline cellulose and displayed reduced activity toward insoluble substrates. A truncated form of the enzyme, in which both the cellulose-binding CBM3b and the fused CBM3c were removed, failed to exhibit significant levels of activity on any of the substrates examined. This study underscores the general nature of this type of enzymatic theme, whereby the fused CBM3c plays a critical accessory role for the family 9 catalytic domain and changes its character to facilitate processive cleavage of recalcitrant cellulose substrates.

Cellulose degradation and assimilation by the unicellular phototrophic eukaryote Chlamydomonas reinhardtii

“…Cellulosic material rather represents an extra carbon source, which has a special relevance under conditions of carbon dioxide limitation. It is not surprising that this system employs cellulases belonging to GHF9, which is the most conserved family of cellulases 43 , comprising endo-as well as exoglucanases, and enzymes of the processive type 44,45 . The ability to degrade soluble (CMC) and crystalline (FP, Avicel) cellulosic substrates to small cellodextrines and CB allows the only conclusion, that the secretome of C. reinhardtii contains an endo-/exoglucanase system or at least one processive endoglucanase.…”

Cellulase, Clostridia, and Ethanol

“…Thus, family IIIc CBDs are better considered as a "cellulose-binding subsite" of the catalytic domain (19,132). Indeed, deletion of the family IIIc CBD from these enzymes rendered the enzyme almost completely inactive (9,90,101,132,285). It is interesting that each cellulosomal catalytic subunit (CelF, CelN, CelG, and EngH) has a family IIIc CBD only but each noncellulosomal enzyme (CelI, CelZ, and E4) has an additional family IIIa CBD.…”

“…However, in some family 9 cellulases, their respective catalytic domains are immediately adjacent to a family IIIc CBD, of which many aromatic amino acid residues on the planar strip thought to be crucial for binding to cellulose are not conserved (19). Examples include CelI (101,123,373), CelN (373), CelQ (9), CelF of C. thermocellum (19), CelG of C. cellulolyticum (90), EngH of C. cellulovorans (194,321), CelZ of C. stercorarium (134), and cellulase E4 of Thermomonospora fusca (132). Family IIIc CBDs are considered to play a very different role from that of the family IIIa CBDs.…”

Family 46 Carbohydrate-binding Modules Contribute to the Enzymatic Hydrolysis of Xyloglucan and β-1,3–1,4-Glucans through Distinct Mechanisms

“…One notable exception to this general trend is CBM3 subfamily c (CBM3c) (19). Members of subfamilies a and b were shown to bind strongly to crystalline cellulose (20,21). In contrast, CBM3cs, as discrete entities, do not bind crystalline cellulosic substrates.…”