During the last 20 years the Animal Nutrition and Biotechnology (ANB) laboratory has been developing cutting edge research to elucidate the role of Carbohydrate-Active enZYmes (CAZYmes) and their ancillary non-catalytic Carbohydrate- Binding Modules (CBMs) in animal nutrition (see highlight 1). In nature these microbial enzymes play a major role in the hydrolysis of complex carbohydrates, such as cellulose and hemicellulose, in the gut of herbivores, thus providing a major source of metabolic energy from dietary fiber. Anaerobic microbes that colonize the gastro intestinal tract of mammals organize CAZymes in high molecular mass multi-enzyme complexes, termed Cellulosomes, which represent one of the most elegant and intricate nanomachines evolved by Nature (see highlight 2). Work developed at ANB laboratory contributed to uncover the mechanism of cellulosome assembly that involves the binding of dockerin domains, located at the C-terminus of cellulases and hemicellulases, to repeated cohesin modules of a structural non-catalytic protein. Thus, highly specific and tight protein:protein interactions modulate the mechanisms of cellulosome organization and cell-surface attachment. Detailed structural, biochemical and molecular studies revealed the intricacies of cellulosomes in different bacteria. More recently these studies culminated in the characterization of a novel mechanism of cellulosome assembly in the major cellulolytic bacterium found in the rumen of mammals, Ruminococcus flavefaciens, which combines more than 200 different enzymes (see highlight 3). Overall these studies indicate that the assembly of CAZymes into cellulosomes potentiates enzyme stability and cooperativity leading to the efficient hydrolysis of recalcitrant polysaccharides. Finally, we have been investigating different biotechnological applications for CAZymes, namely to improve the nutritive value of cereal-based diets for non-herbivore monogastric animals (see highlight 4). During the 2013-2017 period these studies were published in 43 papers and more than 50 protein structures were deposited in the Protein Data Bank (www.rcsb.org/pdb/index.html). A selection of the 10 most exciting papers published by the ANB lab during 2013-2017 is provided below.
Highlights in the period
Highlight 1 – Understanding the role of Family 46 Carbohydrate-binding Modules to the Enzymatic Hydrolysis of different polysaccharides.
In collaboration with the group of Harry Gilbert at The University of Newcastle, UK, ANB lab revealed that the CBM of endo-β-1,4-glucanase B (BhCel5B) from Bacillus halodurans displays a previously unknown function. Strikingly the CBM on its own did not bind soluble or insoluble polysaccharides. The domain was tightly associated with the catalytic module. Depending on the nature of the carbohydrate, the CBM cooperated with the catalytic module to participate in substrate binding or target the enzyme to parts of the cell wall rich in polysaccharides. The data was published in JBC and the paper was selected as 2015 best of JBC papers of the week. BhCel5B structure was selected for the JBC cover (Figure 1).
Highlight 2 – Molecular architecture of Acetivibrio cellulolyticus cellulosome.
Central to cellulosome assembly is a large non-catalytic protein, termed scaffoldin, that contain several repeated cohesin (Coh) domains that establish high affinity protein-protein interactions with dockerin (Doc) modules located in CAZymes. Thus, scaffoldins provide a molecular platform on to which the enzymes will bind through their appended Doc modules. Figure 2 represents the highly complex cellulosome of A. cellulolyticus where several unique adaptor scaffoldins and anchoring scaffoldins contribute to increase the intricacy of this remarkably elaborate nanomachine.
Highlight 3 – Structure of the cohesin-dockerin complex of the cellulosome of the major cellulolytic bacteria in the rumen, Ruminococcus flavefaciens.
In collaboration with groups located at the FCT/UNL (Portugal), The Weismann Institute of Science (Israel), the Universities of Newcastle and York (UK) and the Queen’s University (Canada), ANB lab recently solved the structures of two primordial Coh-Doc complexes in Ruminococcus flavefaciens cellulosome revealing that, in contrast with the majority of these protein-protein interactions identified in nature, R. Docs display a conserved mechanism of Coh recognition involving a single-binding mode (Figure 3). Thus, this work reveals a novel mechanism of cellulosome assembly and challenges the ubiquitous implication of the dual-binding mode in the acquisition of cellulosome flexibility.
Highlight 4 – β-glucanases and not cellulases improve the nutritive value of cereal-based diets for poultry.
Addition of CAZymes to diets containing high levels of soluble non-starch polysaccharides, leads to lower digesta viscosity in poultry and pigs, leading to an improved performance. The ANB lab in collaboration with colleagues at ISA-ULisboa revealed that not all enzymes promote a positive effect in animal nutrition. In the particular case of β-glucan rich diets, β-glucanases but not cellulases, which also effectively cleave the anti-nutritive polysaccharide, improve the nutritive value of cereal-based diets for poultry (Figure 4). High levels of cellulose in diets trap cellulases that become unavailable to attack β-glucans.
1. Bule P, Alves VD, Israeli-Ruimy V, Carvalho AL, Ferreira LM, Smith SP, Gilbert HJ, Najmudin S, Bayer EA & Fontes CMGA (2017). Assembly of Ruminococcus flavefaciens cellulosome revealed by structures of two cohesin-dockerin complexes. Scientific Reports 7:759.
Ruminococcus flavefaciens structures of two group 1 Docs bound to Cohs of primary (ScaA) and adaptor (ScaB) scaffoldins revealed that group 1 Docs display a conserved mechanism of Coh recognition involving a single-binding mode. Thus, this work reveals a novel mechanism of cellulosome assembly and challenges the ubiquitous implication of the dual-binding mode in the acquisition of cellulosome assembly flexibility.
2. Pires VM, Pereira PM, Brás JL, Correia M, Cardoso V, Bule P, Alves VD, Najmudin S, Venditto I, Ferreira LM, Romão MJ, Carvalho AL, Fontes CMGA & Prazeres DM (2017). Stability and ligand promiscuity of type A carbohydrate-binding modules are illustrated by the structure of Spirochaeta thermophila StCBM64C. Journal of Biological Chemistry 292, 4847-4860.
This study details the most extensive carbohydrate-binding platform described so far for a type A CBM, which is able to interact not only with crystalline carbohydrates, but also with diverse solubel polysaccharides. Our results illustate how type A CBm have evolved to increase ligand plasticity and biochemical stability allowing potentiating plant cel wall degradation by complex celulolytic systems.
3. Venditto I, Luis AS, Rydahl M, Schückel J, Fernandes VO, Vidal-Melgosa S, Bule P, Goyal A, Pires VM, Dourado CG, Ferreira LM, Coutinho PM, Henrissat B, Knox JP, Baslé A, Najmudin S, Gilbert HJ, Willats WG & Fontes CMGA (2016). Complexity of the Ruminococcus flavefaciens cellulosome reflects an expansion in glycan recognition. Proceedings of the National Academy of Sciences USA 113, 7136-7141.
This study reveals an unprecedented expansion in glycan recognition by cellulosomes of rumen bacteria specialized in plant cell wall degradation. A description of a unique mechanism by which CBMs modulate enzyme function and how specificity can be tailored by integrating nonspecific catalytic and binding modules into a single enzyme was revealed. Six previously unidentified CBM families that bind β-glucans, β-mannans, and homogalacturonan, four of which were structurally characterized.
4. Bule P, Alves VD, Leitão A, Ferreira LM, Bayer EA, Smith SP, Gilbert HJ, Najmudin S & Fontes CMGA (2016). Single Binding Mode Integration of Hemicellulose-degrading Enzymes via Adaptor Scaffoldins in Ruminococcus flavefaciens Cellulosome. Journal of Biological Chemistry 291, 26658-26669.
R. flavefaciens cellulosomal enzymes, comprising dockerins of groups 3 and 6, predominantly appended to hemicellulose-degrading enzymes, were shown to be indirectly incorporated into primary scaffoldins via an adaptor scaffoldin termed ScaC. This single binding mode interaction with adaptor scaffoldins exemplifies an evolutionary pathway developed by R. flavefaciens to recruit hemicellulases to its cellulosome acting in the gastrointestinal tract of mammals.
5. Brás JL, Pinheiro BA, Cameron K, Cuskin F, Viegas A, Najmudin S, Bule P, Pires VM, Romão MJ, Bayer EA, Spencer HL, Smith S, Gilbert HJ, Alves VD, Carvalho AL & Fontes CMGA (2016). Diverse specificity of cellulosome attachment to the bacterial cell surface. Scientific Reports 6:38292.
Based on the structure and biochemical analysis of five cohesin-dockerin complexes, a dynamic multivalent mode of recruiting cellulosome to the cell surface was elucidated, whereas the cell surface dockerins and its two cohesin-binding interfaces, can present different or identical specificities, maximizing substrate access while facilitating complex assembly.
6. Fernandes VO, Costa M, Ribeiro T, Serrano L, Cardoso V, Santos H, Lordelo M, Ferreira LMA & Fontes CMGA (2016). β-1,3-1,4-glucanases and not β-1,4-glucanases improve the nutritive value of barley-based diets for broilers. Animal Feed Science and Technology 211, 153–163.
The results suggest that in vivo 1,4-β-glucanases tend to act preferentially on cellulosic substrates and not on mixed linked glucans. Although further work is required to extend these observations to other cellulases, the data suggest that exogenous 1,3-1,4-β-glucanases but not 1,4-β-glucanases are obligatory enzymes to improve the nutritive value of barley based diets for broilers.
7. Venditto I, Najmudin S, Luís AS, Ferreira LMA, Sakka K, Knox JP, Gilbert HJ & Fontes CMGA (2015). Family 46 Carbohydrate-binding Modules Contribute to the Enzymatic Hydrolysis of Xyloglucan and β-1,3-1,4-Glucans through Distinct Mechanisms. The Journal of Biological Chemistry 290, 10572-86. (2015 Best JBC Papers of the Week and of the Year).
This report reveals the mechanism by which a CBM can promote enzyme activity through direct interaction with the substrate or by targeting regions of the plant cell wall where the target glucan is abundant., and six previously unidentified CBM families that bind β-glucans, β-mannans, and homogalacturonan, four of which were structurally characterized.
8. Cameron K, Weinstein JY, Zhivin O, Bule P, Fleishman SJ, Alves VD, Gilbert HJ, Ferreira LMA, Fontes CMGA, Bayer EA & Najmudin S (2015). Combined crystal structure of a type-I cohesin, mutation and affinity-binding studies reveal structural determinants of cohesin-dockerin specificity. The Journal of Biological Chemistry 290, 16215-16225.
Combined computational modeling, mutagenesis, and affinity-based binding studies revealed similar hydrogen-bonding networks between putative Ser/Asp recognition residues in the dockerin at positions 11/12 and 45/46, suggesting that a dual-binding mode is not exclusive to the integration of enzymes into primary cellulosomes but can also characterize polycellulosome assembly and cell-surface attachment.
9. Cameron K, Najmudin S, Alves VD, Bayer EA, Smith SP, Bule P, Waller H, Ferreira LM, Gilbert HJ & Fontes CMGA (2015). Cell-surface attachment of bacterial multi-enzyme complexes involves highly dynamic protein-protein anchors. The Journal of Biological Chemistry 290, 13578-13590.
In conjunction with previous studies of the C. thermocellum and C. cellulolyticum cellulosome, this report shows that flexibility in cohesin recognition seems to be a general feature of type I dockerin modules, including those that recruit cellulosomes into the cell surface. Although flexibility resulting from a dual binding mode may not be universal to all cellulosomes, in A. cellulolyticus it seems to be essential not only for enzyme incorporation, but also when the multienzyme complexes are targeted to the cell surface. In this context, the three ScaC cohesins are positioned at the N terminus of the scaffoldin without any detectable linker segment between them.
10. Luís AS, Venditto I, Temple M J, Rogowski A, Baslé A, Xue J, Knox JP, Prates JAM, Ferreira LMA, Fontes CMGA, Najmudin SE & Gilbert HJ (2013). Understanding how noncatalytic carbohydrate binding modules can display specificity for xyloglucan. The Journal of Biological Chemistry 288(7):4799–4809.
This report reveals the mechanism by which β-glucan-specific CBMs can distinguish between linear and mixed linked glucans, and show how these CBMs can exploit an extensive hydrophobic platform to target the side chains of decorated β-glucans.
Over the years the ANB lab has establish a large network of collaborations with scientists in a variety of laboratories, in particular those located in North America (Queen’s University, Canada), the European Union (Universities of York and Newcastle, UK; New University of Lisbon, Portugal), Israel (The Weismann Institute of Science) and India (Indian Institute of Technology Guwahati). These network of collaborations allowed exploring shared infrastructure and human resources and to develop structural, biochemical and molecular studies in a complementary and truly efficient way.
Advanced Studies (PhD e Msc thesis)
During 2013-2017, 6 PhDs were awarded to young scientists developing their studies at the ANB lab, including 2 Marie Curie European PhDs that developed research under the ITN WallTrac (www.walltrac-itn.eu) supported by an EU FP7 grant.
A spin off company was generated from work developed at the ANB lab, NZYTech (www.nzytech.com). Worldwide, NZYTech is the leading company in the provision of a large bank of carbohydrate degrading enzymes for R&D. Currently NZYTech provides >1000 highly purified enzymes in the mg scale. These represent a high value enzyme repository for research and development.