The identification and molecular characterization of the first archaeal bifunctional exo-β-glucosidase/N-acetyl-β-glucosaminidase demonstrate that family GH116 is made of three functionally distinct subfamilies.
BACKGROUND
β-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 β-N-acetylglucosaminidase activities could be involved in the modification/recycling of these cellular components.


METHODS
A thermophilic β-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 β-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 β-glucosidase/β-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.


CONCLUSIONS
The characterization of SSO3039 allows, for the first time in Archaea, the identification of an enzyme involved in the metabolism β-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.
Institute of Protein Biochemistry, Consiglio Nazionale delle Ricerche, Via P. Castellino 111, 80131 Naples, Italy.
Maria Carmina
MC
Ferrara
Beatrice
B
Cobucci-Ponzano
Andrea
A
Carpentieri
Bernard
B
Henrissat
Mosè
M
Rossi
Angela
A
Amoresano
Marco
M
Moracci
D000595:Amino Acid Sequence; D020134:Catalytic Domain; D002853:Chromatography, Liquid; D003001:Cloning, Molecular; D008969:Molecular Sequence Data; D005810:Multigene Family; D010802:Phylogeny; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D048229:Sulfolobus solfataricus / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D053719:Tandem Mass Spectrometry; D001619:beta-N-Acetylhexosaminidases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*
D011994:Recombinant Proteins; D001619:beta-N-Acetylhexosaminidases
'overnight; 2Np-Cel; 2Np-β-cellobioside; 4-Methylumbelliferyl-β-d-glucoside; 4-methylumbelliferyl N-acetyl-β-d-glucosaminide; 4-nitropheny-N-acetyl-β-d-glucosaminide; 4NP-GlcNAc; 4Np-Chit; 4Np-Glc; 4Np-N,N′-diacetyl-β-chitobioside; 4Np-β-glucopyranoside; 5-bromo-4-chloro-indolyl-N-acetyl-β-d-glucosaminide; CAZy; CBE; Carbohydrate active enzymes; FCE; Glc; Glc4; Glc5; GlcNAc; GlcNAc4; GlcNAc5; Glycobiology; HIC; HPAEC-PAD; High Performance Anionic Exchange Chromatography with Pulsed Amperometric Detection; Hydrophobic Interaction Chromatography; MS; MU-Glc; MU-GlcNAc; N-acetylglucosamine; N-butyldeoxynojirimycin; NB-DNJ; Protein N-glycosylation; X-GlcNAc; carbohydrate active enzyme classification; cellopentaose; cellotetraose; conduritol β-epoxide; free cell extract; glucose; mass spectrometry; o.n.; penta-N-acetylchitopentaose; tetra-N-acetylchitotetraose