Ferrara Maria Carmina, Cobucci-Ponzano Beatrice, Carpentieri Andrea, Henrissat Bernard, Rossi Mosè, Amoresano Angela, Moracci Marco
Institute of Protein Biochemistry, Consiglio Nazionale delle Ricerche, Via P. Castellino 111, 80131 Naples, Italy.
Biochim Biophys Acta. 2014 Jan;1840(1):367-77. doi: 10.1016/j.bbagen.2013.09.022. Epub 2013 Sep 21.
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.
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.
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.
This study allowed us to propose that family GH116 is composed of three subfamilies, which show distinct substrate specificities and inhibitor sensitivities.
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.
β-N-乙酰己糖胺酶参与多种生物过程，包括能量代谢、细胞增殖、信号转导以及与病原体相关的炎症和自身免疫性疾病，广泛分布于细菌和真核生物中，但迄今为止在古菌中仅发现少数例子。然而，N-乙酰葡糖胺和半乳糖胺常见于细胞外储存聚合物以及来自不同泉古菌硫化叶菌属丰富表达的糖蛋白上的聚糖中，这表明β-N-乙酰葡糖胺酶活性可能参与这些细胞成分的修饰/再循环。
从嗜热栖热菌P2菌株的细胞提取物中纯化出一种嗜热β-N-乙酰葡糖胺酶，通过质谱鉴定，并在大肠杆菌中克隆和表达。还报道了对不同嗜热栖热菌菌株的糖苷酶测定、稳态动力学常数、底物特异性分析以及重组酶对两种抑制剂的敏感性。
明确鉴定出一种来自嗜热栖热菌的新型β-N-乙酰葡糖胺酶为基因sso3039的产物。详细的酶学表征表明该酶是一种双功能β-葡糖苷酶/β-N-乙酰葡糖胺酶，属于碳水化合物活性酶（CAZy）分类中的GH116家族。
本研究使我们能够提出GH116家族由三个亚家族组成，它们表现出不同的底物特异性和抑制剂敏感性。
SSO3039的表征首次在古菌中鉴定出一种参与β-N-乙酰己糖胺代谢的酶，β-N-乙酰己糖胺是该生命域中糖蛋白的重要组成部分，并大大增加了我们对GH116 CAZy家族成员功能作用和系统发育关系的认识。
