Alderwick Luke J, Seidel Mathias, Sahm Hermann, Besra Gurdyal S, Eggeling Lothar
School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom.
J Biol Chem. 2006 Jun 9;281(23):15653-61. doi: 10.1074/jbc.M600045200. Epub 2006 Apr 4.
The cell wall mycolyl-arabinogalactan-peptidoglycan complex is essential in mycobacterial species, such as Mycobacterium tuberculosis, and is the target of several anti-tubercular drugs. For instance, ethambutol targets arabinogalactan biosynthesis through inhibition of the arabinofuranosyltransferases Mt-EmbA and Mt-EmbB. Following a detailed bioinformatics analysis of genes surrounding the conserved emb locus, we present the identification and characterization of a novel arabinofuranosyltransferase AftA (Rv3792). The enzyme catalyzes the addition of the first key arabinofuranosyl residue from the sugar donor beta-D-arabinofuranosyl-1-monophosphoryldecaprenol to the galactan domain of the cell wall, thus "priming" the galactan for further elaboration by the arabinofuranosyltransferases. Because aftA is an essential gene in M. tuberculosis, we deleted its orthologue in Corynebacterium glutamicum to produce a slow growing but viable mutant. Analysis of its cell wall revealed the complete absence of arabinose resulting in a truncated cell wall structure possessing only a galactan core with a concomitant loss of cell wall-bound mycolates. Complementation of the mutant was fully restored to the wild type phenotype by Cg-aftA. In addition, by developing an in vitro assay using recombinant Escherichia coli expressing Mt-aftA and use of cell wall galactan as an acceptor, we demonstrated the transfer of arabinose from beta-D-arabinofuranosyl-1-monophosphoryldecaprenol to galactan, and unlike the Mt-Emb proteins, Mt-AftA was not inhibited by ethambutol. This newly discovered glycosyltransferase represents an attractive drug target for further exploitation by chemotherapeutic intervention.
细胞壁分枝菌酸 - 阿拉伯半乳聚糖 - 肽聚糖复合物在结核分枝杆菌等分枝杆菌属物种中至关重要，并且是多种抗结核药物的作用靶点。例如，乙胺丁醇通过抑制阿拉伯呋喃糖基转移酶Mt-EmbA和Mt-EmbB来靶向阿拉伯半乳聚糖的生物合成。在对保守的emb基因座周围的基因进行详细的生物信息学分析之后，我们展示了一种新型阿拉伯呋喃糖基转移酶AftA（Rv3792）的鉴定和特性。该酶催化来自糖供体β-D-阿拉伯呋喃糖基-1-单磷酸化癸异戊二烯醇的第一个关键阿拉伯呋喃糖基残基添加到细胞壁的半乳聚糖结构域，从而“启动”半乳聚糖以便由阿拉伯呋喃糖基转移酶进一步修饰。由于aftA是结核分枝杆菌中的必需基因，我们在谷氨酸棒杆菌中删除了其同源物以产生生长缓慢但仍存活的突变体。对其细胞壁的分析显示完全不存在阿拉伯糖，导致形成仅具有半乳聚糖核心的截短细胞壁结构，同时伴随着细胞壁结合的分枝菌酸的丧失。通过Cg-aftA将突变体互补完全恢复到野生型表型。此外，通过开发一种使用表达Mt-aftA的重组大肠杆菌的体外测定法并使用细胞壁半乳聚糖作为受体，我们证明了阿拉伯糖从β-D-阿拉伯呋喃糖基-1-单磷酸化癸异戊二烯醇转移到半乳聚糖，并且与Mt-Emb蛋白不同，Mt-AftA不受乙胺丁醇抑制。这种新发现的糖基转移酶代表了一个有吸引力的药物靶点，可供化疗干预进一步开发利用。
