Protein glycosylation constitutes a critical post-translational modification that supports a vast number of biological functions in living organisms across all domains of life. A seemingly boundless number of enzymes, glycosyltransferases, are involved in the biosynthesis of these protein-linked glycans. Few glycan-biosynthetic glycosyltransferases have been characterized in vitro, mainly due to the majority being integral membrane proteins and the paucity of relevant acceptor substrates. The crenarchaeote Pyrobaculum calidifontis belongs to the TACK superphylum of archaea (Thaumarchaeota, Aigarchaeota, Crenarchaeota, Korarchaeota) that has been proposed as an eukaryotic ancestor. In archaea, N-glycans are mainly found on cell envelope surface-layer proteins, archaeal flagellins and pili. Archaeal N-glycans are distinct from those of eukaryotes, but one noteworthy exception is the high-mannose N-glycan produced by P. calidifontis, which is similar in sugar composition to the eukaryotic counterpart. Here, we present the characterization and crystal structure of the first member of a crenarchaeal membrane glycosyltransferase, PcManGT. We show that the enzyme is a GDP-, dolichylphosphate-, and manganese-dependent mannosyltransferase. The membrane domain of PcManGT includes three transmembrane helices that topologically coincide with “half” of the six-transmembrane helix cellulose-binding tunnel in Rhodobacter spheroides cellulose synthase BcsA. Conceivably, this “half tunnel” would be suitable for binding the dolichylphosphate-linked acceptor substrate. The PcManGT gene (Pcal_0472) is located in a large gene cluster comprising 14 genes of which 6 genes code for glycosyltransferases, and we hypothesize that this cluster may constitute a crenarchaeal N-glycosylation (PNG) gene cluster.

蛋白质糖基化构成了重要的翻译后修饰，可支持生命所有领域中生物体内的大量生物学功能。这些蛋白质连接的聚糖的生物合成中涉及了看似无数的酶，糖基转移酶。很少有糖基生物合成的糖基转移酶在体外得到表征，这主要是由于大多数是完整的膜蛋白和相关受体底物的缺乏。Crenarchaeote Pyrobaculum calidifontis属于古细菌的TACK超级科（Thaumarchaeota，Aigarchaeota，Crenarchaeota，Korarchaeota），已被提出为真核祖先。在古细菌，N-聚糖主要存在于细胞被膜表面层蛋白，古细菌鞭毛蛋白和菌毛上。古细菌的N-聚糖与真核生物不同，但一个值得注意的例外是由P. calidifontis产生的高甘露糖N-聚糖，其糖组成与真核生物类似。在这里，我们介绍了crenarchaeal膜糖基转移酶，Pc ManGT的第一个成员的表征和晶体结构。我们表明该酶是GDP，磷酸二氢磷酸酯和锰依赖的甘露糖基转移酶。Pc ManGT的膜结构域包括三个跨膜螺旋，它们在拓扑上与六个跨膜螺旋纤维素结合通道的“一半”重合。球形红球菌纤维素合成酶BcsA。可以想象，该“半通道”将适用于结合磷酸二硫基酯连接的受体底物。所述PC ManGT基因（Pcal_0472）位于包括14个基因，其6个基因代码糖基转移酶的一个大的基因簇，并且我们假设此群集可以构成crenarchaeal Ñ -glycosylation（PNG）基因簇。