Fitzmaurice A M, Kolattukudy P E
Neurobiotechnology Center and Departments of Biochemistry and Medical Biochemistry, The Ohio State University, Columbus, Ohio 43210, USA.
J Biol Chem. 1998 Apr 3;273(14):8033-9. doi: 10.1074/jbc.273.14.8033.
An open reading frame, ORF3, first identified adjacent to the mycocerosic acid synthase gene in Mycobacterium bovis BCG encodes a protein with acyl-CoA synthase (ACoAS) activity. Genes homologous to acoas are found adjacent to other multifunctional polyketide synthase genes in the mycobacterial genome. To test whether these gene products are necessary to esterify the fatty acids generated by the adjacent polyketide synthase gene products, the acoas gene was disrupted in M. bovis BCG using a suicide vector containing the acoas gene with an internal deletion and the hygromycin-resistant gene as selection marker. Allelic exchange at the acoas locus was confirmed by Southern hybridization and polymerase chain reaction amplification of both flanking regions expected from homologous recombination. Immunoblot analysis indicated that the 65-kDa ACoAS protein product was absent in the mutant. Chromatographic analysis of lipids derived from [1-14C]propionate showed that the mutant did not produce mycocerosyl lipids, although it produced normal levels of mycocerosic acid synthase. These results suggest that ACoAS is involved in the synthesis of mycocerosyl lipids of the mycobacterial cell wall.
一个开放阅读框，即ORF3，最初在卡介苗分枝杆菌的霉菌酸合酶基因附近被鉴定出来，它编码一种具有酰基辅酶A合成酶（ACoAS）活性的蛋白质。在分枝杆菌基因组中，与acoas同源的基因位于其他多功能聚酮合酶基因附近。为了测试这些基因产物对于酯化由相邻聚酮合酶基因产物产生的脂肪酸是否必要，使用一个含有acoas基因内部缺失片段和潮霉素抗性基因作为选择标记的自杀载体，在卡介苗分枝杆菌中破坏了acoas基因。通过Southern杂交以及对同源重组预期的两侧区域进行聚合酶链反应扩增，证实了acoas位点的等位基因交换。免疫印迹分析表明，突变体中不存在65 kDa的ACoAS蛋白产物。对源自[1-14C]丙酸盐的脂质进行色谱分析表明，尽管突变体产生正常水平的霉菌酸合酶，但它不产生霉菌糖脂。这些结果表明，ACoAS参与了分枝杆菌细胞壁霉菌糖脂的合成。
