The molecular characterization of a novel GH38 α-mannosidase from the crenarchaeon Sulfolobus solfataricus revealed its ability in de-mannosylating glycoproteins.

α-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 α-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 α-mannosidase activity depends on a single Zn(2+) ion per subunit is inhibited by swainsonine with an IC(50) of 0.2 mM. The molecular characterization of the native and recombinant enzyme, named Ssα-man, showed that it is highly specific for α-mannosides and α(1,2), α(1,3), and α(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, Ssα-man 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.

beta-Glycosyl azides as substrates for alpha-glycosynthases: preparation of efficient alpha-L-fucosynthases.

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 alpha-glycosynthase. We show here that new mutants of two alpha-L-fucosidases are efficient alpha-L-fucosynthases. The approach shown utilized beta-L-fucopyranosyl azide as donor substrate leading to transglycosylation yields up to 91%. This is the first method exploiting a beta-glycosyl azide donor for alpha-glycosynthases; its applicability to the glycosynthetic methodology in a wider perspective is presented.

The alpha-L-fucosidase from Sulfolobus solfataricus.

Glycoside hydrolases form hyperthermophilic archaea are interesting model systems for the study of catalysis at high temperatures and, at the moment, their detailed enzymological characterization is the only approach to define their role in vivo. Family 29 of glycoside hydrolases classification groups alpha-L-fucosidases involved in a variety of biological events in Bacteria and Eukarya. In Archaea the first alpha-L-fucosidase was identified in Sulfolobus solfataricus as interrupted gene expressed by programmed -1 frameshifting. In this review, we describe the identification of the catalytic residues of the archaeal enzyme, by means of the chemical rescue strategy. The intrinsic stability of the hyperthermophilic enzyme allowed the use of this method, which resulted of general applicability for beta and alpha glycoside hydrolases. In addition, the presence in the active site of the archaeal enzyme of a triad of catalytic residues is a rather uncommon feature among the glycoside hydrolases and suggested that in family 29 slightly different catalytic machineries coexist.

The gene of an archaeal alpha-L-fucosidase is expressed by translational frameshifting.

The standard rules of genetic translational decoding are altered in specific genes by different events that are globally termed recoding. In Archaea recoding has been unequivocally determined so far only for termination codon readthrough events. We study here the mechanism of expression of a gene encoding for a alpha-l-fucosidase from the archaeon Sulfolobus solfataricus (fucA1), which is split in two open reading frames separated by a -1 frameshifting. The expression in Escherichia coli of the wild-type split gene led to the production by frameshifting of full-length polypeptides with an efficiency of 5%. Mutations in the regulatory site where the shift takes place demonstrate that the expression in vivo occurs in a programmed way. Further, we identify a full-length product of fucA1 in S.solfataricus extracts, which translate this gene in vitro by following programmed -1 frameshifting. This is the first experimental demonstration that this kind of recoding is present in Archaea.