Rusnak F, Liu J, Quinn N, Berchtold G A, Walsh C T
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
Biochemistry. 1990 Feb 13;29(6):1425-35. doi: 10.1021/bi00458a013.
The Escherichia coli entB gene, coding for the enterobactin biosynthetic enzyme isochorismatase, has been subcloned into the multicopy plasmid pKK223-3 under the control of the tac promoter. The resulting recombinant plasmid pFR1 expresses isochorismatase amounting to over 50% of the total cellular protein. The enzyme has been purified to homogeneity and a convenient assay developed. The enzyme has a Km for isochorismate of 14.7 microM and a turnover number of 600 min-1. By use of 1H NMR spectroscopy, the progress of the reaction was followed with the expected formation of 2,3-dihydro-2,3-dihydroxybenzoate product. Several substrate analogues were also utilized by the enzyme including chorismic acid, the immediate precursor to isochorismic acid in the enterobactin biosynthetic pathway.
编码肠杆菌素生物合成酶异分支酸酶的大肠杆菌entB基因已被亚克隆到受tac启动子控制的多拷贝质粒pKK223-3中。所得重组质粒pFR1表达的异分支酸酶占细胞总蛋白的50%以上。该酶已被纯化至同质，并开发了一种简便的检测方法。该酶对异分支酸的Km值为14.7微摩尔，周转数为600分钟-1。通过使用1H核磁共振光谱，跟踪反应进程，观察到预期的2,3-二氢-2,3-二羟基苯甲酸产物的形成。该酶还利用了几种底物类似物，包括分支酸，它是肠杆菌素生物合成途径中异分支酸的直接前体。
