Department of Biology, University of Naples Federico II, Naples, Italy.
Institute of Biosciences and BioResources, National Research Council of Italy, Naples, Italy.
Appl Environ Microbiol. 2019 Jan 9;85(2). doi: 10.1128/AEM.01879-18. Print 2019 Jan 15.
is an aerobic crenarchaeal hyperthermophile with optimum growth at temperatures greater than 80°C and pH 2 to 4. Within the crenarchaeal group of , -acetylglucosamine (GlcNAc) has been shown to be a component of exopolysaccharides, forming their biofilms, and of the -glycan decorating some proteins. The metabolism of GlcNAc is still poorly understood in , 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 extracts allowed the detection of a novel α--acetylglucosaminidase (α-GlcNAcase) activity, which has never been identified in Mass spectrometry analysis of the purified activity showed a protein encoded by the gene. Interestingly, the purified recombinant enzyme, which was characterized in detail, revealed a novel de--acetylase activity specific for GlcNAc and derivatives. Thus, assays to identify an α-GlcNAcase found a GlcNAc de--acetylase instead. The α-GlcNAcase activity observed in 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 () suggest the involvement of these enzymes in the GlcNAc metabolism in In this study, a preliminary screening of cellular extracts of allowed the identification of an α--acetylglucosaminidase activity. However, the characterization of the corresponding recombinant enzyme revealed a novel GlcNAc de--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--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 .
是一种需氧的泉古菌超嗜热菌，最适生长温度高于 80°C，pH 值为 2 到 4。在泉古菌组内，N-乙酰氨基葡萄糖（GlcNAc）已被证明是胞外多糖的组成部分，形成其生物膜，并修饰某些蛋白质的β-糖苷。在泉古菌中，GlcNAc 的代谢仍知之甚少，一种获得更多信息的方法是通过鉴定和功能表征参与 GlcNAc 修饰的碳水化合物活性酶（CAZymes）。筛选 提取物检测到一种新型的α-N-乙酰氨基葡萄糖苷酶（α-GlcNAcase）活性，这种活性从未在 中被鉴定过。质谱分析纯化后的活性显示，该活性由 基因编码的蛋白质。有趣的是，经过详细表征的纯化重组酶揭示了一种针对 GlcNAc 和衍生物的新型去乙酰化酶活性。因此，用于鉴定α-GlcNAcase 的测定反而发现了 GlcNAc 的去乙酰化酶。当 SSO2901 与α-葡萄糖苷酶联合使用时，在 提取物中观察到的α-GlcNAcase 活性确实会发生。此外，对基因组上下文的检查和对紧靠 （）上游的假定糖基转移酶的初步表征表明，这些酶参与 中的 GlcNAc 代谢。在这项研究中，对 细胞提取物的初步筛选鉴定了一种α-N-乙酰氨基葡萄糖苷酶活性。然而，对应重组酶的表征揭示了一种新型的 GlcNAc 去乙酰化酶，它与α-葡萄糖苷酶合作，催化 O-α-GlcNAc 糖苷的水解。此外，我们表明，位于去乙酰化酶编码基因侧翼的基因的产物是一种假定的糖基转移酶，表明这两种酶参与了 GlcNAc 的代谢。参与修饰这种必需糖的新型酶活性的发现和功能分析代表了阐明泉古菌生理学和代谢的有力策略。
