Fu H A, Wirt H J, Burris R H, Roberts G P
Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin-Madison 53706.
Gene. 1989 Dec 21;85(1):153-60. doi: 10.1016/0378-1119(89)90475-7.
The function of the cloned draT gene of Rhodospirillum rubrum was studied by placing it under the control of the tac promoter in the vector, pKK223-3. After induction with isopropyl-beta-D-thiogalactopyranoside, dinitrogenase reductase ADP-ribosyltransferase (DRAT) activity was detected in crude extracts of the heterologous hosts Escherichia coli and Klebsiella pneumoniae. In addition, the expression of draT produced a Nif- phenotype in the otherwise wild-type K. pneumoniae strains, the result of the ADP-ribosylation of accumulated dinitrogenase reductase (DR). DR from a nifF- background was also susceptible to ADP-ribosylation, indicating that the oxidized form of DR will serve as a substrate for DRAT in vivo. A mutation that changes the Arg-101 residue of DR, the ADP-ribose attaching site, eliminates the ADP-ribosylation of DR in vivo, confirming the necessity of this residue for modification.
通过将红螺菌（Rhodospirillum rubrum）的克隆draT基因置于载体pKK223 - 3中tac启动子的控制下，对其功能进行了研究。用异丙基 - β - D - 硫代半乳糖苷诱导后，在异源宿主大肠杆菌和肺炎克雷伯菌的粗提取物中检测到了二氮酶还原酶ADP - 核糖基转移酶（DRAT）活性。此外，draT的表达在原本野生型的肺炎克雷伯菌菌株中产生了固氮缺陷型（Nif-）表型，这是积累的二氮酶还原酶（DR）发生ADP - 核糖基化的结果。来自nifF - 背景的DR也易受ADP - 核糖基化作用影响，表明DR的氧化形式在体内将作为DRAT的底物。改变DR的ADP - 核糖附着位点Arg - 101残基的突变消除了DR在体内的ADP - 核糖基化，证实了该残基对于修饰的必要性。
