Leung P, Lee Y M, Greenberg E, Esch K, Boylan S, Preiss J
J Bacteriol. 1986 Jul;167(1):82-8. doi: 10.1128/jb.167.1.82-88.1986.
A mutant strain of Escherichia coli K-12, designated 618, accumulates glycogen at a faster rate than wild-type strain 356. The mutation affects the ADPglucose pyrophosphorylase regulatory properties (N. Creuzat-Sigal, M. Latil-Damotte, J. Cattaneo, and J. Puig, p. 647-680, in R. Piras and H. G. Pontis, ed., Biochemistry of the Glycocide Linkage, 1972). The enzyme is less dependent on the activator, fructose 1,6 bis-phosphate for activity and is less sensitive to inhibition by the inhibitor, 5'-AMP. The structural gene, glgC, for this allosteric mutant enzyme was cloned into the bacterial plasmid pBR322 by inserting the chromosomal DNA at the PstI site. The glycogen biosynthetic genes were selected by cotransformation of the neighboring asd gene into an E. coli mutant also defective in branching enzyme (glgB) activity. Two recombinant plasmids, pEBL1 and pEBL3, that had PstI chromosomal DNA inserts containing glgC and glgB were isolated. Branching enzyme and ADPglucose pyrophosphorylase activities were increased 240- and 40-fold, respectively, in the asd glgB mutant, E. coli K-12 6281. The E. coli K-12 618 mutant glgC gene product was characterized after transformation of an E. coli B ADPglucose pyrophosphorylase mutant with the recombinant plasmid pEBL3. The kinetic properties of the cloned ADPglucose pyrophosphorylase were similar to those of the E. coli K-12 618 enzyme. The inserted DNA in pEBL1 was arranged in opposite orientation to that in pEBL3.
一种名为618的大肠杆菌K - 12突变菌株积累糖原的速度比野生型菌株356更快。该突变影响了ADP葡萄糖焦磷酸化酶的调节特性（N. 克鲁扎特 - 西加尔、M. 拉蒂尔 - 达莫特、J. 卡塔内奥和J. 皮格，第647 - 680页，载于R. 皮拉斯和H. G. 庞蒂斯编，《糖苷键的生物化学》，1972年）。该酶对激活剂1,6 - 二磷酸果糖的活性依赖性降低，对抑制剂5'-AMP的抑制作用也更不敏感。通过将染色体DNA插入PstI位点，将这种别构突变酶的结构基因glgC克隆到细菌质粒pBR322中。通过将相邻的asd基因共转化到一种分支酶（glgB）活性也有缺陷的大肠杆菌突变体中，筛选出糖原生物合成基因。分离出了两个重组质粒pEBL1和pEBL3，它们的PstI染色体DNA插入片段包含glgC和glgB。在asd glgB突变体大肠杆菌K - 12 6281中，分支酶和ADP葡萄糖焦磷酸化酶的活性分别提高了240倍和40倍。在用重组质粒pEBL3转化大肠杆菌B ADP葡萄糖焦磷酸化酶突变体后，对大肠杆菌K - 12 618突变体的glgC基因产物进行了表征。克隆的ADP葡萄糖焦磷酸化酶的动力学特性与大肠杆菌K - 12 618酶的相似。pEBL1中插入的DNA与pEBL3中的方向相反。
