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The Italian National Project of Astrobiology―Life in Space―Origin, Presence, Persistence of Life in Space, from Molecules to Extremophiles A member’s name should be added to the OPPS Project Team: Stavro Ivanovski on page 580 footnotes. We have made this correction on the title page of the article and the eTOC.

Extremophiles are organisms capable of adapt themselves, survive and thrive in hostile habitats that were previously thought to be adverse for life or lethal. Extreme conditions drive the evolution of their inhabitants, highlighting the role of extremophiles as models for the study of the origin and evolution of life on Earth. Indeed, the investigation of both the microbial communities populating these environments and their biocatalysts, as well as of gene expression control by translational recoding in Archaea provide key insights into the boundaries of life, allowing us to speculate mightily about possible extraterrestrial life form. In this framework, we report here recent advances of our research groups in the astrobiology investigation.

In bioeconomy, the development of next generation biorefineries, utilizing energy crops and waste materials, are considered one of the most promising actions toward the sustainable production of fuel and the building blocks for chemicals and polymers with a global market in 2016 of $466.6 billion and a compounded average growth rate of 8.9% between 2016-2021. This complex process is far from being optimized and the application of novel carbohydrate active enzymes justify great expectations. Here we briefly summarise how, for to the advances in (meta)genomics, glycoside hydrolases from (hyper)thermophilic organisms, thanks to their intrinsic stability, can significantly improve lignocellulosic degradation in second generation biorefineries.

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Background ?-N-acetylhexosaminidases, which are involved in a variety of biological processes including energy metabolism, cell proliferation, signal transduction and in pathogen-related inflammation and autoimmune diseases, are widely distributed in Bacteria and Eukaryotes, but only few examples have been found in Archaea so far. However, N-acetylgluco- and galactosamine are commonly found in the extracellular storage polymers and in the glycans decorating abundantly expressed glycoproteins from different Crenarchaeota Sulfolobus sp., suggesting that ?-N-acetylglucosaminidase activities could be involved in the modification/recycling of these cellular components. Methods A thermophilic ?-N-acetylglucosaminidase was purified from cellular extracts of S. solfataricus, strain P2, identified by mass spectrometry, and cloned and expressed in E. coli. Glycosidase assays on different strains of S. solfataricus, steady state kinetic constants, substrate specificity analysis, and the sensitivity to two inhibitors of the recombinant enzyme were also reported. Results A new ?-N-acetylglucosaminidase from S. solfataricus was unequivocally identified as the product of gene sso3039. The detailed enzymatic characterization demonstrates that this enzyme is a bifunctional ?-glucosidase/?-N-acetylglucosaminidase belonging to family GH116 of the carbohydrate active enzyme (CAZy) classification. Conclusions This study allowed us to propose that family GH116 is composed of three subfamilies, which show distinct substrate specificities and inhibitor sensitivities. General significance The characterization of SSO3039 allows, for the first time in Archaea, the identification of an enzyme involved in the metabolism ?-N-acetylhexosaminide, an essential component of glycoproteins in this domain of life, and substantially increases our knowledge on the functional role and phylogenetic relationships amongst the GH116 CAZy family members. © 2013 Elsevier B.V. All rights reserved.

We have recently reported that a functional alpha-L-fucosidase could be expressed by a single insertional mutation in the region of overlap between the ORFs SSO11867 and SSO3060 of the hyperthermophilic Archaeon Sulfolobus solfataricus [Cobucci-Ponzano et al. J. Biol. Chem. (2003) 278, 14622-14631]. This enzyme, belonging to glycoside hydrolase family 29 (GH29), showed micromolar specificity for p-nitrophenyl-alpha-L-fucoside (pNp-Fuc) and promoted transfucosylation reactions by following a reaction mechanism in which the products retained the anomeric configuration of the substrate. The active site residues in GH29 enzymes are still unknown. We describe here the identification of the catalytic nucleophile of the reaction in the alpha-L-fucosidase from S. solfataricus by reactivation with sodium azide of the mutant Asp242Gly that shows a 10(3)-fold activity reduction on pNp-Fuc. The detailed stereochemical analysis of the fucosyl-azide produced by the mutant reactivated on pNp-Fuc revealed its inverted (beta-fucosyl azide) configuration compared with the substrate. This allows for the first time the unambiguous assignment of Asp242, and its homologous residues, as the nucleophilic catalytic residues of GH29 alpha-L-fucosidases. This is the first time that this approach is used for alpha-L-glycosidases, widening the applicability of this method.

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The large-scale production of oligosaccharides is a daunting task, hampering the study of the role of glycans in vivo and the testing of the efficacy of novel glycan-based drugs. Glycosynthases, mutated glycosidases that synthesize oligosaccharides in high yields, are becoming important chemo-enzymatic tools for the production of oligosaccharides. However, while ²-glycosynthase can be produced with a rather well established technology, examples of ±-glycosynthases are thus far limited only to enzymes from families GH29, GH31 and GH95. ±-L-Fucosynthases from GH29 use convenient glycosyl azide derivatives as a strategic alternative to glycosyl fluoride donors. However, the general applicability of this method to other ±-glycosynthases is not trivial and remains to be confirmed. Here, ²-D-galactopyranosyl azide was converted to ±-galacto-oligosaccharides with good yields and high regioselectivity, catalyzed by a novel ±-galactosynthase based on the GH36 ±-galactosidase from the hyperthermophilic bacterium Thermotoga maritima. These results open a new avenue to the practical synthesis of biologically interesting ±-galacto-oligosaccharides and demonstrate more widespread use of ²-glycosyl-azide as donors confirming their utility to expand the repertoire of glycosynthases.

Glycosynthases, engineered glycoside hydrolases that are able to synthesize glycans in quantitative yields without hydrolyzing them, are among the most interesting tools for the chemoenzymatic synthesis of carbohydrates that have been made available so far. From their invention in 1998, these enzymes have been developed enormously and demonstrated to be convenient alternatives to the more expensive glycosyltransferase. Glycosynthases have been proved to be efficient catalysts for the synthesis of oligosaccharides, polysaccharides, and glycoconjugates of various lengths containing different monosaccharides bound through alpha- and beta-linkages, which have interesting applications. In this review we describe the impact that glycosynthases have had on the biocatalysis and biosynthesis of carbohydrates through the description of the novel activities produced and of the development of the glycosynthase-based process. In addition, we summarize the approaches that, according to the current literature, can be adopted to produce novel glycosynthase activities and to improve the existing process

Carbohydrate active enzymes (CAZymes) are a large class of enzymes, which build and breakdown the complex carbohydrates of the cell. On the basis of their amino acid sequences they are classified in families and clans that show conserved catalytic mechanism, structure, and active site residues, but may vary in substrate specificity. We report here the identification and the detailed molecular characterization of a novel glycoside hydrolase encoded from the gene sso1353 of the hyperthermophilic archaeon Sulfolobus solfataricus. This enzyme hydrolyzes aryl beta-gluco- and beta-xylosides and the observation of transxylosylation reactions products demonstrates that SSO1353 operates via a retaining reaction mechanism. The catalytic nucleophile (Glu-335) was identified through trapping of the 2-deoxy-2-fluoroglucosyl enzyme intermediate and subsequent peptide mapping, while the general acid/base was identified as Asp-462 through detailed mechanistic analysis of a mutant at that position, including azide rescue experiments. SSO1353 has detectable homologs of unknown specificity among Archaea, Bacteria, and Eukarya and shows distant similarity to the non-lysosomal bile acid beta-glucosidase GBA2 also known as glucocerebrosidase. On the basis of our findings we propose that SSO1353 and its homologs are classified in a new CAZy family, named GH116, which so far includes beta-glucosidases (EC 3.2.1.21), beta-xylosidases (EC 3.2.1.37), and glucocerebrosidases (EC 3.2.1.45) as known enzyme activities.