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International audience; Pompe disease is an inherited metabolic disorder due to the deficiency of the lysosomal acid α-glucosidase (GAA). The only approved treatment is enzyme replacement therapy with the recombinant enzyme (rhGAA). Further approaches like pharmacological chaperone therapy, based on the stabilising effect induced by small molecules on the target enzyme, could be a promising strategy. However, most known chaperones could be limited by their potential inhibitory effects on patient's enzymes. Here we report on the discovery of novel chaperones for rhGAA, L- and D-carnitine, and the related compound acetyl-D-carnitine. These drugs stabilise the enzyme at pH and temperature without inhibiting the activity and acted synergistically with active-site directed pharmacological chaperones. Remarkably, they enhanced by 4-fold the acid α-glucosidase activity in fibroblasts from three Pompe patients with added rhGAA. This synergistic effect of L-carnitine and rhGAA has the potential to be translated into improved therapeutic efficacy of ERT in Pompe disease.

Genetic code decoding, initially considered to be universal and immutable, is now known to be flexible. In fact, in specific genes, ribosomes deviate from the standard translational rules in a programmed way, a phenomenon globally termed recoding. Translational recoding, which has been found in all domains of life, includes a group of events occurring during gene translation, namely stop codon readthrough, programmed ± 1 frameshifting, and ribosome bypassing. These events regulate protein expression at translational level and their mechanisms are well known and characterized in viruses, bacteria and eukaryotes. In this review we summarize the current state-of-the-art of recoding in the third domain of life. In Archaea, it was demonstrated and extensively studied that translational recoding regulates the decoding of the 21st and the 22nd amino acids selenocysteine and pyrrolysine, respectively, and only one case of programmed –1 frameshifting has been reported so far in Saccharolobus solfataricus P2. However, further putative events of translational recoding have been hypothesized in other archaeal species, but not extensively studied and confirmed yet. Although this phenomenon could have some implication for the physiology and adaptation of life in extreme environments, this field is still underexplored and genes whose expression could be regulated by recoding are still poorly characterized. The study of these recoding episodes in Archaea is urgently needed.

In the field of biocatalysis and the development of a bio-based economy, hemicellulases have attracted great interest for various applications in industrial processes. However, the study of the catalytic activity of the lignocellulose-degrading enzymes needs to be improved to achieve the efficient hydrolysis of plant biomasses. In this framework, hemicellulases from hyperthermophilic archaea show interesting features as biocatalysts and provide many advantages in industrial applications thanks to their stability in the harsh conditions encountered during the pretreatment process. However, the hemicellulases from archaea are less studied compared to their bacterial counterpart, and the activity of most of them has been barely tested on natural substrates. Here, we investigated the hydrolysis of xyloglucan oligosaccharides from two different plants by using, both synergistically and individually, three glycoside hydrolases from Saccharolobus solfataricus: a GH1 β-gluco-/β-galactosidase, a α-fucosidase belonging to GH29, and a α-xylosidase from GH31. The results showed that the three enzymes were able to release monosaccharides from xyloglucan oligosaccharides after incubation at 65 °C. The concerted actions of β-gluco-/β-galactosidase and the α-xylosidase on both xyloglucan oligosaccharides have been observed, while the α-fucosidase was capable of releasing all α-linked fucose units from xyloglucan from apple pomace, representing the first GH29 enzyme belonging to subfamily A that is active on xyloglucan.

Understanding the formation of biogenic molecules in abiotic conditions is a prerequisite in the origin-of-life studies. Determining the conditions allowing an efficient one-pot synthesis of the largest possible panel of biogenic compounds may shed light on the plausible scenario in which the processes that kick-started life might have occurred. We report a set of experiments describing the syntheses taking place from formamide (NH2CHO) and thermal water in the presence of meteorites, in the hydrothermal physical-chemical environment of the Phlegrean Fields. The results show that meteorites catalyse the synthesis of a large panel of organic compounds of biological relevance, including carboxylic acids, nucleobases, amino acids and sugars. The simplicity of the system (a one-carbon molecule as starting compound, a volcanic hydrothermal environment, meteorites as catalysts) hints to a possible extension of the results to similar environments present in other planetary bodies and space objects.

The highly stereocontrolled de novo synthesis of L-NBDNJ (the unnatural enantiomer of the iminosugar drug Miglustat) and a preliminary evaluation of its chaperoning potential are herein reported. L-NBDNJ is able to enhance lysosomal α-glucosidase levels in Pompe disease fibroblasts, either when administered singularly or when co-incubated with the recombinant human α-glucosidase. In addition, differently from its D-enantiomer, L-NBDNJ does not act as a glycosidase inhibitor.

Chemo-enzymatic synthesis of oligosaccharides exploits the diversity of glycosidases and their ability to promote transglycosylation reactions in parallel with hydrolysis. Methods to increase the transglycosylation/hydrolysis ratio include site-directed mutagenesis and medium modification. The former approach was successful in several cases and has provided the best synthetic yields with glycosynthases-mutants at the catalytic nucleophile position that promote transglycosylation with high efficiency, but do not hydrolyze the oligosaccharide products. Several glycosidases have proven recalcitrant to this conversion, thus alternative methods to increase the transglycosylation/hydrolysis ratio by mutation would be very useful. Here we show that a mutant of a β-galactosidase from Alicyclobacillus acidocaldarius in an invariant residue in the active site of the enzymes of this family (glutamic acid 361) carries out efficient transglycosylation reactions on different acceptors only in the presence of external ions with yields up to 177-fold higher than that of the wild type. This is the first case in which sodium azide and sodium formate in combination with site-directed mutagenesis have been used to introduce transglycosylation activity into a glycosidase. These observations will hopefully guide further efforts to generate useful synthases.

Sulfolobus solfataricus is an aerobic crenarchaeal hyperthermophile with optimum growth at temperatures greater than 80°C and pH 2 to 4. Within the crenarchaeal group of Sulfolobales, N-acetylglucosamine (GlcNAc) has been shown to be a component of exopolysaccharides, forming their biofilms, and of the N-glycan decorating some proteins. The metabolism of GlcNAc is still poorly understood in Archaea, and one approach to gaining additional information is through the identification and functional characterization of carbohydrate active enzymes (CAZymes) involved in the modification of GlcNAc. The screening of S. solfataricus extracts allowed the detection of a novel α-N-acetylglucosaminidase (α-GlcNAcase) activity, which has never been identified in Archaea. Mass spectrometry analysis of the purified activity showed a protein encoded by the sso2901 gene. Interestingly, the purified recombinant enzyme, which was characterized in detail, revealed a novel de-N-acetylase activity specific for GlcNAc and derivatives. Thus, assays to identify an α-GlcNAcase found a GlcNAc de-N-acetylase instead. The α-GlcNAcase activity observed in S. solfataricus extracts did occur when SSO2901 was used in combination with an α-glucosidase. Furthermore, the inspection of the genomic context and the preliminary characterization of a putative glycosyltransferase immediately upstream of sso2901 (sso2900) suggest the involvement of these enzymes in the GlcNAc metabolism in S. solfataricus. IMPORTANCE In this study, a preliminary screening of cellular extracts of S. solfataricus allowed the identification of an α-N-acetylglucosaminidase activity. However, the characterization of the corresponding recombinant enzyme revealed a novel GlcNAc de-N-acetylase, which, in cooperation with the α-glucosidase, catalyzed the hydrolysis of O-α-GlcNAc glycosides. In addition, we show that the product of a gene flanking the one encoding the de-N-acetylase is a putative glycosyltransferase, suggesting the involvement of the two enzymes in the metabolism of GlcNAc. The discovery and functional analysis of novel enzymatic activities involved in the modification of this essential sugar represent a powerful strategy to shed light on the physiology and metabolism of Archaea.

In the framework of the discovery of new thermophilic enzymes of potential biotechnological interest, we embarked in the characterization of a new thermophilic esterase from the thermophilic bacterium Geobacillus thermodenitrificans. The phylogenetic analysis of the GTNG_0744 esterase indicated that the sequence belongs to the enterochelin/enterobactin esterase group, which have never been recognized as a family in the lipases/esterase classification. These enzymes catalyze the last step in the acquisition of environmental Fe3+ through siderophore hydrolysis. In silico analysis revealed, for the first time, that the machinery for the uptake of siderophores is present in G. thermodenitrificans. The purified recombinant enzyme, EstGtA3, showed different substrate specificity from known enterochelin/enterobactin esterases, recognizing short chain esters with a higher specificity constant for 4-NP caprylate. The enzyme does not require cofactors for its activity, is active in the pH range 7.0–8.5, has highest activity at 60 °C and is 100% stable when incubated for 16 h at 55 °C. DTT, β-mercaptoethanol and Triton X-100 have an activating effect on the enzymatic activity. Organic solvents have in general a negative effect on the enzyme, but n-hexane is a strong activator up to 150, making EstGtA3 a good candidate for applications in biotechnology.

Background The Bacillus subtilis spore has long been used to display antigens and enzymes. Spore display can be accomplished by a recombinant and a non-recombinant approach, with the latter proved more efficient than the recombinant one. We used the non-recombinant approach to independently adsorb two thermophilic enzymes, GH10-XA, an endo-1,4-β-xylanase (EC 3.2.1.8) from Alicyclobacillus acidocaldarius, and GH3-XT, a β-xylosidase (EC 3.2.1.37) from Thermotoga thermarum. These enzymes catalyze, respectively, the endohydrolysis of (1-4)-β-d-xylosidic linkages of xylans and the hydrolysis of (1-4)-β-d-xylans to remove successive d-xylose residues from the non-reducing termini. Results We report that both purified enzymes were independently adsorbed on purified spores of B. subtilis. The adsorption was tight and both enzymes retained part of their specific activity. When spores displaying either GH10-XA or GH3-XT were mixed together, xylan was hydrolysed more efficiently than by a mixture of the two free, not spore-adsorbed, enzymes. The high total activity of the spore-bound enzymes is most likely due to a stabilization of the enzymes that, upon adsorption on the spore, remained active at the reaction conditions for longer than the free enzymes. Spore-adsorbed enzymes, collected after the two-step reaction and incubated with fresh substrate, were still active and able to continue xylan degradation. The recycling of the mixed spore-bound enzymes allowed a strong increase of xylan degradation. Conclusion Our results indicate that the two-step degradation of xylans can be accomplished by mixing spores displaying either one of two required enzymes. The two-step process occurs more efficiently than with the two un-adsorbed, free enzymes and adsorbed spores can be reused for at least one other reaction round. The efficiency of the process, the reusability of the adsorbed enzymes, and the well documented robustness of spores of B. subtilis indicate the spore as a suitable platform to display enzymes for single as well as multi-step reactions.

We report here on the preparation of a novel ?-thioglycoligase that can be used for the fast and efficient synthesis of ?-N-acetylglucosamine-based glycosides. Using the ?-N-acetyl-glucosaminidase from Clostridium perfringens of family GH89 (according to the Carbohydrate Active Enzymes classification) as starting point, we prepared mutants in the acid/base residue glutamic acid 483 that were found to have different synthetic efficiencies (maximal yields >80% after 24 h) in the presence of an activated donor and suitable acceptors. The synthetic potential of the Glu483 alanine mutant as an ?-thioglycoligase - only the third biocatalyst with this stereospecificity reported to date, and the first selective for the N-acetylglucosamine moiety - was demonstrated by producing for the first time N-acetyl-?-d-glucosaminyl azide and N-acetylglucosamine ?-thioglycosides in high yields. To showcase the application of such compounds, we show that they offer the wild-type CpGH89 protection from thermal unfolding, demonstrating their potential as pharmacological chaperones for the treatment of mucopolysaccharidosis IIIB (Sanfilippo syndrome). (Figure presented.).

Pompe disease, a rare lysosomal storage disease caused by deficiency of the lysosomal acid α-glucosidase (GAA), is characterized by glycogen accumulation, triggering severe secondary cellular damage and resulting in progressive motor handicap and premature death. Numerous disease-causing mutations in the gaa gene have been reported, but the structural effects of the pathological variants were unknown. Here we present the high-resolution crystal structures of recombinant human GAA (rhGAA), the standard care of Pompe disease. These structures portray the unbound form of rhGAA and complexes thereof with active site-directed inhibitors, providing insight into substrate recognition and the molecular framework for the rationalization of the deleterious effects of disease-causing mutations. Furthermore, we report the structure of rhGAA in complex with the allosteric pharmacological chaperone N-acetylcysteine, which reveals the stabilizing function of this chaperone at the structural level., Pompe disease is caused by mutations in lysosomal acid α-glucosidase (GAA) and patients are being treated with recombinant human α-glucosidase (rhGAA). Here the authors present the crystal structures of rhGAA and its complexes with inhibitors and a pharmacological chaperone, which is important for drug development.

Translational recoding includes a group of events occurring during gene translation, namely stop codon readthrough, programmed +/- 1 frameshifting, and ribosome bypassing, which have been found in organisms from all domains of life. They serve to regulate protein expression at translational level and represent a relatively less known exception to the traditional central 'dogma' of biology that information flows as DNA -> RNA -> protein and that it is stored in a co-linear way between the 5'-> 3' of nucleic acids and N -> C-terminal of polypeptides. In archaea, in which translational recoding regulates the decoding of the 21st and the 22nd amino acids selenocysteine and pyrrolysine, respectively, only one case of programmed -1 frameshifting has been reported so far and further examples, although promising, have not been confirmed yet. We here summarize the current state-of-the-art of this field that, especially in archaea, has relevant implications for the physiology of life in extreme environments and for the origin of life.

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 β-gluco- and β-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 β-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 β-glucosidases (EC 3.2.1.21), β-xylosidases (EC 3.2.1.37), and glucocerebrosidases (EC 3.2.1.45) as known enzyme activities.

Glycosynthases are mutant glycosidases, which in the presence of activated glycosides and suitable reaction conditions, synthesize oligosaccharides without hydrolysing them. This feature makes these catalysts promising tools for the large scale synthesis of carbohydrates. However, despite the popularity of the glycosynthetic approach, the number of enzymes effecting glycosynthetic reactions is still limited. We report here on the design of novel reaction conditions for a thermophilic α-l-fucosidase mutant, which might provide a route for the production of novel glycosynthases.

Background Biofuels production from plant biomasses is a complex multi-step process with important economic burdens. Several biotechnological approaches have been pursued to reduce biofuels production costs. The aim of the present study was to explore the production in tobacco plastome of three genes encoding (hemi)cellulolytic enzymes from thermophilic and hyperthermophilic bacterium and Archaea, respectively, and test their application in the bioconversion of an important industrially pretreated biomass feedstock (A. donax) for production of second-generation biofuels. Results The selected enzymes, endoglucanase, endo-β-1,4-xylanase and β-glucosidase, were expressed in tobacco plastome with a protein yield range from 2 % to more than 75 % of total soluble proteins (TSP). The accumulation of endoglucanase (up to 2 % TSP) gave altered plant phenotypes whose severity was directly linked to the enzyme yield. The most severe seedling-lethal phenotype was due to the impairment of plastid development associated to the binding of endoglucanase protein to thylakoids. Endo-β-1,4-xylanase and β-glucosidase, produced at very high level without detrimental effects on plant development, were enriched (fourfold) by heat treatment (105.4 and 255.4 U/mg, respectively). Both plastid-derived biocatalysts retained the main features of the native or recombinantly expressed enzymes with interesting differences. Plastid-derived xylanase and β-glucosidase resulted more thermophilic than the E. coli recombinant and native counterpart, respectively. Bioconversion experiments, carried out at 50 and 60 °C, demonstrated that plastid-derived enzymes were able to hydrolyse an industrially pretreated giant reed biomass. In particular, the replacement of commercial enzyme with plastid-derived xylanase, at 60 °C, produced an increase of both xylose recovery and hydrolysis rate; whereas the replacement of both xylanase and β-glucosidase produced glucose levels similar to those observed with the commercial cocktails, and xylose yields always higher in the whole 24–72 h range. Conclusions The very high production level of thermophilic and hyperthermophilic enzymes, their stability and bioconversion efficiencies described in this study demonstrate that plastid transformation represents a real cost-effective production platform for cellulolytic enzymes. Electronic supplementary material The online version of this article (doi:10.1186/s13068-016-0569-z) contains supplementary material, which is available to authorized users.

Summary Standard decoding of the genetic information into polypeptides is performed by one of the most sophisticated cell machineries, the translating ribo- some, which, by following the genetic code, ensures the correspondence between the mature mRNA and the protein sequence. However, the expression of a minority of genes requires programmed deviations from the standard decoding rules, globally named recoding . This includes ribosome programmed -/ + 1 frameshifting, ribosome hopping, and stop codon readthrough. Recoding in Archaea was unequivo- cally demonstrated only for the translation of the UGA stop codon into the amino acid selenocysteine. However, a new recoding event leading to the 22nd amino acid pyrrolysine and the preliminary reports on a gene regulated by programmed - 1 frameshifting have been recently described in Archaea. Therefore, it appears that the study of this phenomenon in Archaea is still at its dawn and that most of the genes whose expression is regulated by recoding are still uncharacterized.

Carbohydrates serve as structural components and en- ergy sources of cells. More interestingly, however, these biomolecules are involved in a variety of molecular recognition processes in intercellular communication and signal transduction such as cell adhesion, differ- entiation, development and regulation. For these rea- sons, great interest has arisen in carbohydrate-based pharmaceuticals and on the development of techniques for the analysis and synthesis of oligosaccharides. In this respect, enzymes involved in carbohydrates hydrolysis and modification are increasingly being utilised for the bioconversion of sugars, for the synthesis of oligosac- charides with potential application, and for the charac- terisation of carbohydrate compounds of unknown structure. In this review, the enzymology and the applications of three glycosyl hydrolases from the archaeon Sulfolobus solfataricus are described. In particular, we focus on the enzymological properties of a b-glycosidase, an a-xylo- sidase, and an a-fucosidase; their exploitation in oligo- saccharides synthesis will be also described.

The biotransformation of lignocellulose biomasses into fermentable sugars is a very complex procedure including, as one of the most critical steps, the (hemi) cellulose hydrolysis by specific enzymatic cocktails. We explored here, the potential of stable glycoside hydrolases from thermophilic organisms, so far not used in commercial enzymatic preparations, for the conversion of glucuronoxylan, the major hemicellulose of several energy crops. Searches in the genomes of thermophilic bacteria led to the identification, efficient production, and detailed characterization of novel xylanase and a-glucuronidase from Alicyclobacillus acidocaldarius (G1-110-XA and GH67-GA, respectively) and a alpha-glucuronidase from Caldicellulosiruptor saccharolyticus (GH67-GC). Remarkably, GH10-XA, if compared to other thermophilic xylanases from this family, coupled good specificity on beechwood xylan and the best stability at 65 degrees C (3.5 days). In addition, GH67-GC was the most stable alpha-glucuronidases from this family and the first able to hydrolyse both aldouronic acid and aryl-alpha-glucuronic acid substrates. These enzymes, led to the very efficient hydrolysis of beechwood xylan by using 7- to 9-fold less protein (concentrations

The first, recently identified, archaeal alpha-xylosidase from Sulfolobus solfataricus (XylS) shows high specificity for hydrolysis of isoprimeverose [alpha-D-xylopyranosyl-(1,6)-D-glucopyranose, (X)], the p-nitrophenyl-beta derivative of isoprimeverose, and xyloglucan oligosaccharides and has transxylosidic activity, forming, in a retaining mode, interesting alpha-xylosides. This article describes the synthesis of isoprimeverose, the disaccharidic repeating unit of xyloglucan, of the p-nitrophenyl-beta derivative of isoprimeverose, and of a trisaccharide based on isoprimeverose that is one of the trisaccharidic building blocks of xyloglucan. A substrate structure-activity relationship is recognized for both the hydrolysis and the synthesis reactions of XylS, it being a biocatalyst (i) active hydrolytically only on X-ending substrates liberating a xylose molecule and (ii) capable of transferring xylose only on the nonreducing end glucose of p-nitrophenyl-(PNP)-beta-D-cellobioside. The compounds synthesized by this enzyme are a starting point for enzymological studies of other new enzymes (i.e., xyloglucanases) for which suitable substrates are difficult to synthesize. This study also allows us to define the chemical characteristics of the xylose-transferring activity of this new archaeal enzyme, contributing to building up a library of different glycosidases with high specific selectivity for oligosaccharide synthesis.

We here report the first molecular characterization of an alpha-xylosidase (XylS) from an Archaeon. Sulfolobus solfataricus is able to grow at temperatures higher than 80 degrees C on several carbohydrates at acidic pH. The isolated xylS gene encodes a monomeric enzyme homologous to alpha-glucosidases, alpha-xylosidases, glucoamylases and sucrase-isomaltases of the glycosyl hydrolase family 31. xylS belongs to a cluster of four genes in the S. solfataricus genome, including a beta-glycosidase, an hypothetical membrane protein homologous to the major facilitator superfamily of transporters, and an open reading frame of unknown function. The alpha-xylosidase was overexpressed in Escherichia coli showing optimal activity at 90 degrees C and a half-life at this temperature of 38 h. The purified enzyme follows a retaining mechanism of substrate hydrolysis, showing high hydrolytic activity on the disaccharide isoprimeverose and catalyzing the release of xylose from xyloglucan oligosaccharides. Synergy is observed in the concerted in vitro hydrolysis of xyloglucan oligosaccharides by the alpha-xylosidase and the beta-glycosidase from S. solfataricus. The analysis of the total S. solfataricus RNA revealed that all the genes of the cluster are actively transcribed and that xylS and orf3 genes are cotranscribed.

The second generation bioethanol represents a main challenge in global efforts to utilize renewable resources rather than fossil fuels. However, the close association of cellulose and hemicelluloses to lignin in the plant cell wall makes it difficult to degrade lignocellulose into fermentable sugars. Consequently, pretreatments are necessary to make the polysaccharides more accessible to the enzymes, but the high temperature and extreme pH conditions required give rise to problems when using conventional enzymes in the saccharification step (Galbe and Zacchi 2002). Microorganisms thriving in habitats characterized by harsh conditions, and the enzymes derived therein, represent a helpful tool in the development of bioethanol production processes. In fact, they allow bioconversions at non-conventional conditions under which common biocatalysts are denatured. The use of high operational temperatures allows energy savings by reducing the cooling cost after high temperature pretreatments, and, in ethanol production, thermophilic conditions permit ethanol evaporation allowing harvest during fermentation.

Pompe disease (PD) is a metabolic myopathy due to the deficiency of the lysosomal enzyme alpha-glucosidase (GAA). The only approved treatment for this disorder, enzyme replacement with recombinant human GAA (rhGAA), has shown limited therapeutic efficacy in some PD patients. Pharmacological chaperone therapy (PCT), either alone or in combination with enzyme replacement, has been proposed as an alternative therapeutic strategy. However, the chaperones identified so far also are active site-directed molecules and potential inhibitors of target enzymes. We demonstrated that N-acetylcysteine (NAC) is a novel allosteric chaperone for GAA. NAC improved the stability of rhGAA as a function of pH and temperature without disrupting its catalytic activity. A computational analysis of NAC-GAA interactions confirmed that NAC does not interact with GAA catalytic domain. NAC enhanced the residual activity of mutated GAA in cultured PD fibroblasts and in COS7 cells overexpressing mutated GAA. NAC also enhanced rhGAA efficacy in PD fibroblasts. In cells incubated with NAC and rhGAA, GAA activities were 3.7-8.7-fold higher than those obtained in cells treated with rhGAA alone. In a PD mouse model the combination of NAC and rhGAA resulted in better correction of enzyme activity in liver, heart, diaphragm and gastrocnemia, compared to rhGAA alone. Received 12 May 2012; accepted 3 July 2012; advance online publication 18 September 2012. doi:10.1038/mt.2012.152

Glycosynthases are engineered glycoside hydrolases that in suitable reaction conditions promote the synthesis of oligosaccharides with exquisite stereoselectivity and enhanced regioselectivity, if compared to traditional chemical methods. This approach was demonstrated to be successful in a number of cases including β-glycosynthases acting at the termini or within an oligosaccharide chain ( exo - and endo -glycosynthases, respectively) and, more recently, α-glycosynthases. This led to the production of a vast repertoire of products that include poly- and oligosaccharides, glycoconjugates, and glycopeptides. These molecules can be used as ligands of glycoside hydrolases, for the characterization of therapeutic enzymes, and as leads of drugs for the pharmaceutical industry. In this panorama, hyperthermophilic organisms, which thrive at temperatures as high as 80 °C, which usually impede the growth of other living forms, have been used in the development of interesting novel glycosynthases. In fact, the extreme stability of these catalysts to extremes of pH and high concentrations of organics has allowed the exploration of novel reaction conditions, revealing new avenues for enzyme-catalyzed oligosaccharide synthesis.

Covering: up to March 2012 Oligo-, polysaccharides, and glycoconjugates are a relevant part of the bioactive components of the natural products exploited in therapeutics, diagnostics, food additives, and biomaterials. Glycans are directly involved in important biological processes, such as immunostimulation, anti-inflammatory, antioxidant, and chemoprotectant actions and/or are crucial for their activity, by modulating target recognition, stability, and pharmacokinetics. On the other hand, carbohydrate extracts used for functional studies are rather heterogeneous and lack structural information because of their intrinsic complexity hampering purification and characterization. Therefore, methods for glycoside synthesis and modification are urgently needed. Recently, glycosynthases, engineered glycoside hydrolases with no hydrolytic activity that synthesize glycans in quantitative yields, were introduced. Here we will illustrate how the glycosynthases described so far might be exploited for the production of glycan analogs of natural products and their enormous potential in this field.

International audience; An interesting approach for the chemo-enzymatic synthesis of carbohydrates is the use of glycosynthases, a class of mutant glycosidases derived from β-glycoside hydrolases obtained by replacement of the active site nucleophile with a non-nucleophilic residue. However, the scarcity of α-glycosynthases has so far hampered access to the synthesis of a large class of oligosaccharides of biotechnological interest. We review here a new glycosynthetic methodology for the production of two retaining α-fucosynthases and an α-galactosynthase exploiting β-glycosyl azide derivatives. The general applicability of this approach, which opens new perspectives in the use of azide derivatives for the production of novel α-glycosynthases, is discussed.

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 α- and β-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.

Glycosidases, the enzymes responsible in nature for the catabolism of carbohydrates, are well-studied catalysts widely used in industrial biotransformations and oligosaccharide synthesis, which are also attractive targets for drug development. Glycosidases from hyperthermophilic organisms (thriving at temperatures > 85 degrees C) are also interesting models to understand the molecular basis of protein stability and to produce robust tools for industrial applications. Here, we review the results obtained in the last two decades by our group on a beta-glycosidase from the hyperthermophilic Archaeon Sulfolobus solfataricus. Our findings will be presented in the general context of the stability of proteins from hyperthermophiles and of the chemo-enzymatic synthesis of oligosaccharides.

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 beta-glycosynthase can be produced with a rather well-established technology, examples of alpha-glycosynthases are thus far limited only to enzymes from glycoside hydrolase 29 (GH29), GH31 and GH95 families. alpha-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 alpha-glycosynthases is not trivial and remains to be confirmed. Here, beta-d-galactopyranosyl azide was converted to alpha-galacto-oligosaccharides with good yields and high regioselectivity, catalyzed by a novel alpha-galactosynthase based on the GH36 alpha-galactosidase from the hyperthermophilic bacterium Thermotoga maritima. These results open a new avenue to the practical synthesis of biologically interesting alpha-galacto-oligosaccharides and demonstrate more widespread use of beta-glycosyl-azide as donors, confirming their utility to expand the repertoire of glycosynthases.

alpha-Mannosidases, important enzymes in the N-glycan processing and degradation in Eukaryotes, are frequently found in the genome of Bacteria and Archaea in which their function is still largely unknown. The a-mannosidase from the hyperthermophilic Crenarchaeon Sulfolobus solfataricus has been identified and purified from cellular extracts and its gene has been cloned and expressed in Escherichia coli. The gene, belonging to retaining GH38 mannosidases of the carbohydrate active enzyme classification, is abundantly expressed in this Archaeon. The purified alpha-mannosidase activity depends on a single Zn2+ ion per subunit is inhibited by swainsonine with an IC50 of 0.2 mM. The molecular characterization of the native and recombinant enzyme, named Ss alpha-man, showed that it is highly specific for alpha-mannosides and alpha(1,2), alpha(1,3), and alpha(1,6)-D-mannobioses. In addition, the enzyme is able to demannosylate Man(3)GlcNAc(2) and Man(7)GlcNAc(2) oligosaccharides commonly found in N-glycosylated proteins. More interestingly, Ss alpha-man removes removes mannose residues from the glycosidic moiety of the bovine pancreatic ribonuclease B, suggesting that it could process mannosylated proteins also in vivo. This is the first evidence that archaeal glycosidases are involved in the direct modification of glycoproteins. (C) 2010 Elsevier Masson SAS. All rights reserved.

We have previously shown that the major ion-pairs network of the tetrameric beta-glycosidase from the hyperthermophilic archeon Sulfolobus solfataricus involves more than 16 ion-pairs and hydrogen bonds between several residues from the four subunits and protects the protein from thermal unfolding by sewing the carboxy-termini of the enzyme. We show here that the amino-terminal of the enzyme also plays a relevant role in the thermostabilization of the protein. In fact, the addition of four extra amino acids at the amino-terminal of the beta-glycosidase, though not affecting the catalytic machinery of the enzyme and its thermophilicity, produced a faster enzyme inactivation in the temperature range 85-95 degrees C and decreased the Tm of the protein of 6 degrees C, measured by infrared spectroscopy. In addition, detailed two-dimensional IR correlation analysis revealed that the quaternary structure of the tagged enzyme is destabilized at 85 degrees C whilst that of the wild type enzyme is stable up to 98 degrees C. Molecular models allowed the rationalization of the experimental data indicating that the longer amino-terminal tail may destabilize the beta-glycosidase by enhancing the molecular fraying of the polypeptide and loosening the dimeric interfaces. The data support the hypothesis that fraying of the polypeptide chain termini is a relevant event in protein unfolding.

Retaining glycosidases promote the hydrolysis of the substrate by following a double-displacement mechanism involving a covalent intermediate. The catalytic residues are a general acid/base catalyst and the nucleophile. Experimental identification of these residues in a specific glycosidase allows for the assigning of the corresponding residues in all of the other enzymes belonging to the same family. By means of sequence alignment, mutagenesis, and detailed kinetic studies of the alpha-fucosidase from Sulfolobus solfataricus (Ssalpha-fuc) (family 29), we show here that the residues, invariant in this family, have the function inferred from the analysis of the 3D structure of the enzyme from Thermotoga maritima (Tmalpha-fuc). These include in Ssalpha-fuc the substrate-binding residues His46 and His123 and the nucleophile of the reaction, previously described. The acid/base catalyst could be assigned less easily. The k(cat) of the Ssalpha-fucGlu292Gly mutant, corresponding to the acid/base catalyst of Tmalpha-fuc, is reduced by 154-fold but could not be chemically rescued. Instead, the Ssalpha-fucGlu58Gly mutant revealed a 4000-fold reduction of k(cat)/K(M) if compared to the wild-type and showed the rescue of the k(cat) by sodium azide at wild-type levels. Thus, our data suggest that a catalytic triad, namely, Glu58, Glu292, and Asp242, is involved in catalysis. Glu58 and Glu292 cooperate in the role of acid/base catalyst, while Asp242 is the nucleophile of the reaction. Our data suggest that in glycosidase family 29 alpha-fucosidases promoting the retaining mechanism with slightly different catalytic machineries coexist.

In oligosaccharide synthesis the chemo-en- zymatic approach is often chosen to obtain the de- sired glycosidic bond by exploiting the exquisite sub- strate specificity and stereoselectivity of glycosidases and glycosyltransferases. In recent years the invention of new enzymatic activities, named glycosynthases, created a third route for the enzymatic synthesis of oligosaccharides. Glycosynthases are mutant glycosi- dases, devoid of hydrolytic activity, that produce oli- gosaccharides with yields > 80%. The glycosynthase technology is now well established and was greatly improved recently. Several groups experimentally proved that this methodology can be applied to sever- al glycosidases and that the activity of existing glyco- synthases can be significantly enhanced. In this review we briefly describe the characteristics of these syn- thetic tools and show a survey of the most recent find- ings. Methods for the prediction of the glycosynthetic potential of known glycosidases are also discussed.

Alpha-L-Fucosidase is a lysosomal enzyme responsible for hydrolyzing the alpha-1,6-linked fucose joined to the reducing-end N-acetylglucosamine of carbohydrate moieties in glycoproteins. The first alpha-l-fucosidase from Archaea was recently identified in the genome of the hyperthermophile Sulfolobus solfataricus; the enzyme is encoded by two open reading frames separated by a -1 frameshift. A preliminary biochemical and biophysical characterization of this extremophile enzyme has been carried out both in solution, through small angle X-ray scattering experiments, and in the crystalline state, showing an unusual oligomeric assembly resulting from the association of nine subunits, endowed with 3-fold molecular symmetry.

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.

Biochemical, crystallographic, and computational data support the hypothesis that electrostatic interactions are among the dominant forces in stabilizing hyperthermophilic proteins. The thermostable β-glycosidase from the hyperthermophile Sulfolobus solfataricus (Ssβ-gly) is an interesting model system for the study of protein adaptation to high temperatures. The largest ion-pair network of Ssβ-gly is located at the tetrameric interface of the molecule; in this paper, key residues in this region were modified by site-directed mutagenesis and the stability of the mutants was analyzed by kinetics of thermal denaturation. All mutations produced faster enzyme inactivation, suggesting that the C-terminal ionic network prevents the dissociation into monomers, which is the limiting step in the mechanism of Ssβ-gly inactivation. Moreover, the calculated reaction order showed that the mechanism of inactivation depends on the mutation introduced, suggesting that intermediates maintaining enzymatic activity are produced during the inactivation transition of some, but not all, mutants. Molecular models of each mutant allow us to rationalize the experimental evidence and give support to the current theories on the mechanism of ion pair stabilization in proteins from hyperthermophiles. Proteins 2002;48:98–106. © 2002 Wiley-Liss, Inc.

International audience; Fucose-containing oligosaccharides play a central role in physio-pathological events, and fucosylated oligosaccharides have interesting potential applications in biomedicine. No methods for the large-scale production of oligosaccharides are currently available, but the chemo-enzymatic approach is very promising. Glycosynthases, mutated glycosidases that synthesize oligosaccharides in high yields, have been demonstrated to be an interesting alternative. However, examples of glycosynthases available so far are restricted to a limited number of glycosidases families and to only one retaining a-glycosynthase. We show here that new mutants of two a-L-fucosidases are efficient a-L-fucosynthases. The approach shown utilized b-L-fucopyranosyl azide as donor substrate leading to transglycosylation yields up to 91%. This is the first method exploiting a b-glycosyl azide donor for a-glycosynthases; its applicability to the glycosynthetic methodology in a wider perspective is presented.

S beta gly and CelB are well-studied hyperthermophilic glycosyl hydrolases, isolated from the Archaea Sulfolobus solfataricus and Pyrococcus furiosus, respectively. Previous studies revealed that the two enzymes are phylogenetically related; they are very active and stable at high temperatures, and their overall three-dimensional structure is very well conserved. To acquire insight in the molecular determinants of thermostability and thermoactivity of these enzymes, we have performed a detailed comparison, under identical conditions, of enzymological and biochemical parameters of S beta gly and CelB, and we have probed the basis of their stability by perturbations induced by temperature, pH, ionic strength, and detergents. The major result of the present study is that, although the two enzymes are remarkably similar with respect to kinetic parameters, substrate specificity, and reaction mechanism, they are strikingly different in stability to the different physical or chemical perturbations induced. These results provide useful information for the design of further experiments aimed at understanding the structure-function relationships in these enzymes.