Division of Molecular Microbiology, University of Dundee, Dundee DD1 5EH, UK.
Open Biol. 2013 Sep 11;3(9):130022. doi: 10.1098/rsob.130022.
The fungal cell possesses an essential carbohydrate cell wall. The outer layer, mannan, is formed by mannoproteins carrying highly mannosylated O- and N-linked glycans. Yeast mannan biosynthesis is initiated by a Golgi-located complex (M-Pol I) of two GT-62 mannosyltransferases, Mnn9p and Van1p, that are conserved in fungal pathogens. Saccharomyces cerevisiae and Candida albicans mnn9 knockouts show an aberrant cell wall and increased antibiotic sensitivity, suggesting the enzyme is a potential drug target. Here, we present the structure of ScMnn9 in complex with GDP and Mn(2+), defining the fold and catalytic machinery of the GT-62 family. Compared with distantly related GT-78/GT-15 enzymes, ScMnn9 carries an unusual extension. Using a novel enzyme assay and site-directed mutagenesis, we identify conserved amino acids essential for ScMnn9 'priming' α-1,6-mannosyltransferase activity. Strikingly, both the presence of the ScMnn9 protein and its product, but not ScMnn9 catalytic activity, are required to activate subsequent ScVan1 processive α-1,6-mannosyltransferase activity in the M-Pol I complex. These results reveal the molecular basis of mannan synthesis and will aid development of inhibitors targeting this process.
真菌细胞具有必需的碳水化合物细胞壁。外层甘露聚糖由携带高度甘露糖基化 O-和 N-连接聚糖的甘露糖蛋白组成。酵母甘露聚糖的生物合成由位于高尔基体的复合物 (M-Pol I) 启动，该复合物由两种 GT-62 甘露糖基转移酶 Mnn9p 和 Van1p 组成，这两种酶在真菌病原体中保守。酿酒酵母和白色念珠菌 mnn9 敲除突变体表现出异常的细胞壁和增加的抗生素敏感性，表明该酶是一个潜在的药物靶点。在这里，我们展示了 ScMnn9 与 GDP 和 Mn(2+) 复合物的结构，定义了 GT-62 家族的折叠和催化机制。与远缘相关的 GT-78/GT-15 酶相比，ScMnn9 携带一个不寻常的延伸。使用新型酶测定和定点突变，我们确定了 ScMnn9“引发”α-1,6-甘露糖基转移酶活性所必需的保守氨基酸。引人注目的是，无论是 ScMnn9 蛋白的存在还是其产物，而不是 ScMnn9 催化活性，都需要在 M-Pol I 复合物中激活随后的 ScVan1 连续α-1,6-甘露糖基转移酶活性。这些结果揭示了甘露聚糖合成的分子基础，并将有助于开发针对该过程的抑制剂。
