Govezensky D, Greener T, Segal G, Zamir A
Biochemistry Department, Weizmann Institute of Science, Rehovot, Israel.
J Bacteriol. 1991 Oct;173(20):6339-46. doi: 10.1128/jb.173.20.6339-6346.1991.
Several approaches were used to study the role of GroEL, the prototype chaperonin, in the nitrogen fixation (nif) system. An Escherichia coli groEL mutant transformed with the Klebsiella pneumoniae nif gene cluster accumulated very low to nondetectable levels of nitrogenase components compared with the isogenic wild-type strain or the mutant cotransformed with the wild-type groE operon. In K. pneumoniae, overexpression of the E. coli groE operon markedly accelerated the rate of appearance of the MoFe protein and its constituent polypeptides after the start of derepression. The groEL mutation in E. coli decreased NifA-dependent beta-galactosidase expression from the nifH promoter but did not affect the constitutive expression of nifA from the tet promoter of ntr-controlled expression from the nifLA promoter. The possibility that GroEL is required for the correct folding of NifA was supported by coimmunoprecipitation of NifA with anti-GroEL antibodies. Kinetic analyses of nitrogenase assembly in 35S pulse-chased K. pneumoniae pointed to the existence of high-molecular-weight intermediates in MoFe protein assembly and demonstrated the transient binding of newly synthesized NifH and NifDK to GroEL. Overall, these results indicate that GroEL fulfills both regulatory and structural functions in the nif system.
采用了几种方法来研究伴侣蛋白GroEL（伴侣蛋白的原型）在固氮（nif）系统中的作用。与同基因野生型菌株或与野生型groE操纵子共转化的突变体相比，用肺炎克雷伯菌nif基因簇转化的大肠杆菌groEL突变体积累的固氮酶成分水平非常低，甚至检测不到。在肺炎克雷伯菌中，大肠杆菌groE操纵子的过表达在去阻遏开始后显著加速了钼铁蛋白及其组成多肽的出现速率。大肠杆菌中的groEL突变降低了nifH启动子的NifA依赖性β-半乳糖苷酶表达，但不影响ntr控制的nifLA启动子的tet启动子对nifA的组成型表达。NifA与抗GroEL抗体的共免疫沉淀支持了GroEL是NifA正确折叠所必需的这一可能性。对35S脉冲追踪的肺炎克雷伯菌中固氮酶组装的动力学分析表明，钼铁蛋白组装过程中存在高分子量中间体，并证明新合成的NifH和NifDK与GroEL存在瞬时结合。总体而言，这些结果表明GroEL在nif系统中兼具调节和结构功能。
