Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80523-1682, USA.
Glycobiology. 2009 Nov;19(11):1235-47. doi: 10.1093/glycob/cwp116. Epub 2009 Aug 4.
Arabinogalactan (AG) and lipoarabinomannan (LAM) are the two major cell wall (lipo)polysaccharides of mycobacteria. They share arabinan chains made of linear segments of alpha-1,5-linked D-Araf residues with some alpha-1,3-branching, the biosynthesis of which offers opportunities for new chemotherapeutics. In search of the missing arabinofuranosyltransferases (AraTs) responsible for the formation of the arabinan domains of AG and LAM in Mycobacterium tuberculosis, we identified Rv0236c (AftD) as a putative membrane-associated polyprenyl-dependent glycosyltransferase. AftD is 1400 amino acid-long, making it the largest predicted glycosyltransferase of its class in the M. tuberculosis genome. Assays using cell-free extracts from recombinant Mycobacterium smegmatis and Corynebacterium glutamicum strains expressing different levels of aftD indicated that this gene encodes a functional AraT with alpha-1,3-branching activity on linear alpha-1,5-linked neoglycolipid acceptors in vitro. The disruption of aftD in M. smegmatis resulted in cell death and a decrease in its activity caused defects in cell division, reduced growth, alteration of colonial morphology, and accumulation of trehalose dimycolates in the cell envelope. Overexpression of aftD in M. smegmatis, in contrast, induced the accumulation of two arabinosylated compounds with carbohydrate backbones reminiscent of that of LAM and a degree of arabinosylation dependent on aftD expression levels. Altogether, our results thus indicate that AftD is an essential AraT involved in the synthesis of the arabinan domain of major mycobacterial cell envelope (lipo)polysaccharides.
阿拉伯半乳聚糖（AG）和脂阿拉伯甘露聚糖（LAM）是分枝杆菌细胞壁（脂）多糖的两种主要成分。它们都含有阿拉伯聚糖链，由线性的α-1,5 连接的 D-Araf 残基组成，具有一些α-1,3 分支，其生物合成为新的化学疗法提供了机会。在寻找负责分枝杆菌结核分枝杆菌 AG 和 LAM 阿拉伯聚糖结构域形成的缺失阿拉伯呋喃糖基转移酶（AraTs）时，我们鉴定出 Rv0236c（AftD）为一种假定的膜相关多萜醇依赖性糖基转移酶。AftD 长 1400 个氨基酸，使其成为结核分枝杆菌基因组中该类最大的预测糖基转移酶。使用来自重组分枝杆菌和谷氨酸棒状杆菌菌株的无细胞提取物进行的测定表明，该基因编码一种功能性 AraT，具有在体外线性α-1,5 连接的新糖脂受体上的α-1,3 分支活性。在分枝杆菌 smegmatis 中敲除 aftD 会导致细胞死亡，并降低其活性，从而导致细胞分裂缺陷、生长减少、菌落形态改变以及细胞包膜中双分枝海藻糖二霉素的积累。相比之下，在分枝杆菌 smegmatis 中过表达 aftD 会诱导两种阿拉伯糖基化化合物的积累，其碳水化合物骨架类似于 LAM，并且阿拉伯糖基化程度取决于 aftD 的表达水平。总的来说，我们的结果表明 AftD 是参与主要分枝杆菌细胞壁（脂）多糖阿拉伯聚糖结构域合成的必需 AraT。
