The hemicellulose-degrading enzyme system of the thermophilic bacterium Clostridium stercorarium: comparative characterisation and addition of new hemicellulolytic glycoside hydrolases

Background: The bioconversion of lignocellulosic biomass in various industrial processes, such as the production of biofuels, requires the degradation of hemicellulose. Clostridium stercorarium is a thermophilic bacterium, well known for its outstanding hemicellulose-degrading capability. Its genome comprises about 50 genes for partially still uncharacterised thermostable hemicellulolytic enzymes. These are promising candidates for industrial applications. Results: To reveal the hemicellulose-degrading potential of 50 glycoside hydrolases, they were recombinantly produced and characterised. 46 of them were identified in the secretome of C. stercorarium cultivated on cellobiose. Xylanases Xyn11A, Xyn10B, Xyn10C, and cellulase Cel9Z were among the most abundant proteins. The secretome of C. stercorarium was active on xylan, beta-glucan, xyloglucan, galactan, and glucomannan. In addition, the recombinant enzymes hydrolysed arabinan, mannan, and galactomannan. 20 enzymes are newly described, degrading xylan, galactan, arabinan, mannan, and aryl-glycosides of beta-D-xylose, beta-D-glucose, beta-D-galactose, alpha-L-arabinofuranose, alpha-L-rhamnose, beta-D-glucuronic acid, and N-acetyl-beta-D-glucosamine. The activities of three enzymes with non-classified glycoside hydrolase (GH) family modules were determined. Xylanase Xyn105F and beta-D-xylosidase Bxl31D showed activities not described so far for their GH families. 11 of the 13 polysaccharide-degrading enzymes were most active at pH 5.0 to pH 6.5 and at temperatures of 57-76 degrees C. Investigation of the substrate and product specificity of arabinoxylan-degrading enzymes revealed that only the GH10 xylanases were able to degrade arabinoxylooligosaccharides. While Xyn10C was inhibited by alpha-(1,2)-arabinosylations, Xyn10D showed a degradation pattern different to Xyn10B and Xyn10C. Xyn11A released longer degradation products than Xyn10B. Both tested arabinose-releasing enzymes, Arf51B and Axh43A, were able to hydrolyse single-as well as double-arabinosylated xylooligosaccharides. Conclusions: The obtained results lead to a better understanding of the hemicellulose-degrading capacity of C. stercorarium and its involved enzyme systems. Despite similar average activities measured by depolymerisation tests, a closer look revealed distinctive differences in the activities and specificities within an enzyme class. This may lead to synergistic effects and influence the enzyme choice for biotechnological applications. The newly characterised glycoside hydrolases can now serve as components of an enzyme platform for industrial applications in order to reconstitute synthetic enzyme systems for complete and optimised degradation of defined polysaccharides and hemicellulose

Overexpression of Q-rich prion-like proteins suppresses polyQ cytotoxicity and alters the polyQ interactome

Expansion of a poly-glutamine (polyQ) repeat in a group of functionally unrelated proteins is the cause of several inherited neurodegenerative disorders, including Huntington's disease. The polyQ length-dependent aggregation and toxicity of these disease proteins can be reproduced in Saccharomyces cerevisiae. This system allowed us to screen for genes that when overexpressed reduce the toxic effects of an N-terminal fragment of mutant huntingtin with 103 Q. Surprisingly, among the identified suppressors were three proteins with Q-rich, prion-like domains (PrDs): glycine threonine serine repeat protein (Gts1p), nuclear polyadenylated RNA-binding protein 3, and minichromosome maintenance protein 1. Overexpression of the PrD of Gts1p, containing an imperfect 28 residue glutamine-alanine repeat, was sufficient for suppression of toxicity. Association with this discontinuous polyQ domain did not prevent 103Q aggregation, but altered the physical properties of the aggregates, most likely early in the assembly pathway, as reflected in their increased SDS solubility. Molecular simulations suggested that Gts1p arrests the aggregation of polyQ molecules at the level of nonfibrillar species, acting as a cap that destabilizes intermediates on path to form large fibrils. Quantitative proteomic analysis of polyQ interactors showed that expression of Gts1p reduced the interaction between polyQ and other prion-like proteins, and enhanced the association of molecular chaperones with the aggregates. These findings demonstrate that short, Q-rich peptides are able to shield the interactive surfaces of toxic forms of polyQ proteins and direct them into nontoxic aggregates