Comparative characterization of all cellulosomal cellulases from Clostridium thermocellum reveals high diversity in endoglucanase product formation essential for complex activity

Abstract

Background: Clostridium thermocellum is a paradigm for efficient cellulose degradation and a promising organism for the production of second generation biofuels. It owes its high degradation rate on cellulosic substrates to the presence of supra-molecular cellulase complexes, cellulosomes, which comprise over 70 different single enzymes assembled on protein-backbone molecules of the scaffold protein CipA.

Results: Although all 24 single-cellulosomal cellulases were described previously, we present the first comparative catalogue of all these enzymes together with a comprehensive analysis under identical experimental conditions, including enzyme activity, binding characteristics, substrate specificity, and product analysis. In the course of our study, we encountered four types of distinct enzymatic hydrolysis modes denoted by substrate specificity and hydrolysis product formation: (i) exo-mode cellobiohydrolases (CBH), (ii) endo-mode cellulases with no specific hydrolysis pattern, endoglucanases (EG), (iii) processive endoglucanases with cellotetraose as intermediate product (pEG4), and (iv) processive endoglucanases with cellobiose as the main product (pEG2). These modes are shown on amorphous cellulose and on model cello-oligosaccharides (with degree of polymerization DP 3 to 6). Artificial mini-cellulosomes carrying combinations of cellulases showed their highest activity when all four endoglucanase-groups were incorporated into a single complex. Such a modeled nonavalent complex (n = 9 enzymes bound to the recombinant scaffolding protein CipA) reached half of the activity of the native cellulosome. Comparative analysis of the protein architecture and structure revealed characteristics that play a role in product formation and enzyme processivity.

Conclusions: The identification of a new endoglucanase type expands the list of known cellulase functions present in the cellulosome. Our study shows that the variety of processivities in the enzyme complex is a key enabler of its high cellulolytic efficiency. The observed synergistic effect may pave the way for a better understanding of the enzymatic interactions and the design of more active lignocellulose-degrading cellulase cocktails in the future.

Keywords: Biomass degradation; Cellobiohydrolase; Cellulase; Cellulosome; Endoglucanase; Molecular docking; Synergistic hydrolysis; Synthetic complex.

Fig. 1

Comparison of all β-1,4-glucanases from the C. thermocellum cellulosome (listed in first column). Second column: Intermediate and final product analysis of different cellulases on various cello-oligosaccharides and PASC as substrate. The arrays show the oligosaccharide products (degree of polymerization ranging from glucose DP 1 to cellohexaose DP 6) on the Y-axis and the kinetic product shift over time (X-axis). The sugar amount detected by thin-layer chromatography is depicted as heat map representation, with relative intensities of the sugar products ranging from 1% (light gray) to 100% (black). Explanations of time points: 0.5 = 0.5 min; 2 = 2 min; 5 = 5 min; ¼ h = 15 min; 1 h = 60 min; 2 h = 120 min; on = overnight incubation. Empty fields (white) indicate that no products were formed, or products were below the detection limit of thin-layer chromatography. The pattern of protein CtCel124 is not shown due to its low activity. Third column: Activity of recombinant cellulases on various substrates (average values from triplicate measurements) at optimal cellulosome activity parameters (60 °C, pH 5.8; see Additional file 6). Fourth column: presence of glycoside hydrolase (GH) families, carbohydrate-binding modules (CBM), and Ig-like modules (Ig). For continuation of this figure, please see Fig. 2

Fig. 2

Comparison of all β-1,4-glucanases from the C. thermocellum cellulosome (continuation of Fig. 1)

Fig. 3

Hydrolytic efficiency of multi-enzyme complexes on Avicel as substrate. a End-point activity of 1 µg of the enzymes on 0.25% Avicel after 42 h in dependence of the endoglucanase functions present in the complex. The bars represent amount of reducing sugar ends (glucose equivalents) as average values from biological replicates (at least duplicate measurements with standard deviations represented as × 1 SD). The endoglucanase product pattern present (+) or absent (−) in the complex was non-processive (EG) or processive with cellobiose (pEG2) or cellotetraose (pEG4) as intermediate or main product. The complex “all EG types” consists of 9 different enzymes, whereas each cellulase function is present in this complex (cellobiohydrolases, non-processive endoglucanases, members of pEG2 and pEG4, respectively). Each of the complexed mixtures comprised equal stoichiometric loading and statistical distribution of eight single enzymes on CipA8 by cohesin–dockerin protein interaction. The enzyme complexes were purified by gel filtration to exclude the impact of unbound single cellulases. As controls, the activity of complexed and non-complexed enzyme extracts of C. thermocellum mutant SM901 [41] is shown together with the native cellulosome. Abbreviations: Cel8A (A), Cel9D (D), Cel5-26H (H), Cel9-44J (J), Cel9K (K), Cel5L (L), Cel9R (R), and Cel48S (S). b Enzyme kinetics of cellulosomal complexes on 2.5% Avicel. c Electrophoretic mobility shift showing the binding capacity of recombinant scaffolding protein CipA8 made possible by its eight cohesin binding modules. Complex formation by cohesin–dockerin interaction is visible by up-shifted protein bands in the native gel. 10 µM of CipA8 was titrated with 80 µM of a nonavalent cellulase mixture (all EG types + CipA8) for statistically binding all free cohesin modules. As another control, the SM901 enzyme extract was also completely bound (SM901 + CipA8). The 6% native PAGE gel was stained with Coomassie R-250

Fig. 4

Structure-based multiple sequence alignment of GH9 family catalytic modules of four C. thermocellum cellulases: Cel9D, Cbh9A, Cel9T, and Cel9F. α-Helices (α- and η-helices), β-sheets, and loops in Cbh9A are indicated and numbered above the sequences as squiggles and arrows, respectively. Strict α-turns are indicated with TTT, strict β-turns with TT. The catalytic triad in the active sites is indicated with asterisks. Amino acids of the endoglucanase TfCel9A from Thermobifida fusca known to be involved in substrate-binding [59, 60] are shown as black triangles, those identified from cellobiohydrolase Cbh9A [56] are marked as gray triangles. The numbers below indicate the corresponding cello-oligosaccharide positions reported to interact/bind. Carbohydrate positions + 1 and + 2 are the expected product sites. Loop regions conferring exo-activity of Cbh9A are highlighted in light blue [56]

Fig. 5

Structural comparison of four different catalytic clefts from glycoside hydrolase GH9 cellulases. The figure depicts cellobiohydrolase Cbh9A (PDB structure 1RQ5), processive endoglucanase pEG2 (Cel9D, PDB 1CLC), pEG4 (Cel9F, predicted structure), and non-processive endoglucanase Cel9T (PDB accession 2YIK) as gray surface plots and their corresponding sugar-binding moieties (red sticks) and catalytic triad (in blue). Cellohexaose (Glc₆) was taken from PDB 7CEL. Numbers in black depict cello-oligosaccharide positions (+ 2 to − 2) according to the nomenclature for sugar-binding subsites [61] in the catalytic cleft from protein–ligand interaction data for Cbh9A according to [56], Cel9T [57], Cel9A from T. fusca [59] and the structural sequence alignment (Fig. 4)