Interplay between Clostridium thermocellum family 48 and family 9 cellulases in cellulosomal versus noncellulosomal states

Abstract

The anaerobic, thermophilic cellulolytic bacterium Clostridium thermocellum is known for its elaborate cellulosome complex, but it also produces a separate free cellulase system. Among the free enzymes, the noncellulosomal enzyme Cel9I is a processive endoglucanase whose sequence and architecture are very similar to those of the cellulosomal enzyme Cel9R; likewise, the noncellulosomal exoglucanase Cel48Y is analogous to the principal cellulosomal enzyme Cel48S. In this study we used the designer cellulosome approach to examine the interplay of prominent cellulosomal and noncellulosomal cellulases from C. thermocellum. Toward this end, we converted the cellulosomal enzymes to noncellulosomal chimeras by swapping the dockerin module of the cellulosomal enzymes with a carbohydrate-binding module from the free enzyme analogues and vice versa. This enabled us to study the importance of the targeting effect of the free enzymes due to their carbohydrate-binding module and the proximity effect for cellulases on the designer cellulosome. C. thermocellum is the only cellulosome-producing bacterium known to express two different glycoside hydrolase family 48 enzymes and thus the only bacterial system that can currently be used for such studies. The different activities with crystalline cellulose were examined, and the results demonstrated that the individual chimeric cellulases were essentially equivalent to the corresponding wild-type analogues. The wild-type cellulases displayed a synergism of about 1.5-fold; the cellulosomal pair acted synergistically when they were converted into free enzymes, whereas the free enzymes acted synergistically mainly in the wild-type state. The targeting effect was found to be the major factor responsible for the elevated activity observed for these specific enzyme combinations, whereas the proximity effect appeared to play a negligible role.

FIG. 1.

Schematic diagrams of the wild-type enzymes, chimeric enzymes, and chimeric scaffoldins that were used in this study. The modular notation, structure, and molecular mass of each protein are indicated. White indicates a C. thermocellum cellulosome-derived component; black indicates a C. thermocellum noncellulosomal component; and gray indicates a B. cellulosolvens cellulosome-derived component. In the modular notation of the enzymes, the numbers indicate the family of the catalytic domain; R, I, S, and Y indicate the original names of the enzymes (Cel9R, Cel9I, Cel48S, and Cel48Y, respectively); and b and t indicate the source of the dockerin module (B. cellulosolvens from ScaB and C. thermocellum from Cel48S, respectively). B and T indicate the source of the divergent cohesins (B, the first cohesin from ScaA of B. cellulosolvens; T, the third cohesin from CipA of C. thermocellum). An asterisk indicates a converted enzyme (an enzyme converted either from cellulosomal to noncellulosomal, in which the native dockerin was replaced by a cellulose-binding CBM, or vice versa).

FIG. 2.

Comparative degradation of Avicel by individual family 48 and family 9 enzymes. Cellulosomal enzymes were converted to the free mode, and free enzymes were converted to the cellulosomal mode. The compositions of the reaction mixtures in this figure and Fig. 3 and 4 are indicated by diagrams above the bars. A white structure indicates a cellulosome-derived enzyme, and a black structure indicates a non-cellulosome-derived enzyme. Open bars, 48S and 9R catalytic modules in either “WT-cellulosomal,” “mock-free” or “converted-free” mode; filled bars, 48Y-CBM and 9I-CBM catalytic modules in either the “WT-free,” “converted-cellulosomal,” or “mock-free” mode. For definitions of enzyme systems see Table 2. The results of two independent experiments are shown. Triplicate reactions were carried out, and standard deviations are indicated by error bars. The relative activity was determined by comparison with the activity of the WT-free enzyme 9I-CBM, as described in Materials and Methods.

FIG. 3.

Comparative degradation of Avicel by combinations of family 48 and family 9 enzymes. Open bars, conversion from cellulosomal mode to free mode (the cellulosomal enzyme 48S and 9R catalytic modules in “WT-cellulosomal,” “mock-free,” and “designer-WT” modes were converted into the “converted-free” mode); filled bars, conversion from free mode to cellulosomal mode (the free 48Y and 9I catalytic modules in a “WT-free” mode were converted into a cellulosomal mode, either “converted-cellulosomal,” “mock-free,” or “designer-converted”). For definitions of enzyme systems see Table 2. The results of two independent experiments are shown. Triplicate reactions were carried out, and standard deviations are indicated by error bars. The relative activity was determined by comparison with the activity of the WT-free enzymes 48Y-CBM and 9I-CBM, as described in Materials and Methods.

FIG. 4.

Synergism between combinations of family 48 and family 9 enzymes. (Left side) Synergy of the cellulosomal enzymes 48S and 9R for catalytic modules in “WT-cellulosomal,” “mock-free,” or “designer-WT” mode and converted into “converted-free” mode. The open bars indicate the calculated sums of activities of the individual enzymes; the contribution of the family 48 enzyme (top section) is considerably less than that of the family 9 enzyme (bottom section) for each of the pairs. The striped bars indicate the observed activity experimentally obtained for the combined enzyme systems. (Right side) Synergy of the free enzymes 48Y and 9I for catalytic modules in a “WT-free” mode and converted into cellulosomal modes, including “converted-cellulosomal,” “mock-free,” and “designer-converted.” The solid filled bars indicate the calculated sums of activities of the individual enzymes (top section, family 48 enzyme; bottom section, family 9 enzyme). For definitions of enzyme systems see Table 2. Values for single enzyme activities and pairs of enzymatic activities are shown in Table S3 in the supplemental material. Triplicate reactions were carried out. The relative activity was determined by comparison with the activity of the WT-free enzymes 48Y-CBM and 9I-CBM, as described in Materials and Methods.