The FixL protein of Rhizobium meliloti can be separated into a heme-binding oxygen-sensing domain and a functional C-terminal kinase domain.
The FixL protein of Rhizobium meliloti can be separated into a heme-binding oxygen-sensing domain and a functional C-terminal kinase domain.
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DOI：
10.1073/pnas.89.10.4280
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发表时间：
1992-05
影响因子：
11.1
通讯作者：
E. Monson;M. Weinstein;G. Ditta;D. Helinski
中科院分区：
文献类型：
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作者：
E. Monson;M. Weinstein;G. Ditta;D. Helinski
苜蓿根瘤菌中固氮（nif 和 fix）基因的转录是由氧浓度降低诱导的。两个基因fixL和fixJ的产物负责感知和传递低氧信号。由fixL和fixJ（分别为FixL和FixJ）编码的蛋白质与通过常见磷酸转移机制转导环境信号的细菌蛋白质家族同源[David, M., Daveran, M., Batut, J., Dedieu, A., Domergue, O., Ghai, J., Hertig, C., Boistard, P. & Khan, D. (1988) Cell 54, 671-683]。 FixL，氧传感器，是一种膜蛋白。先前已表明 FixL 的可溶性衍生物 FixL* 是一种氧结合血红素蛋白，也是一种自磷酸化和磷酸化 FixJ 的激酶 [Gilles-Gonzalez, M. A., Ditta, G. S. & Helinski, D. R. (1991) Nature (London) 350, 170-172]。在这项工作中，构建了fixL*的缺失衍生物，并在大肠杆菌中过表达，并纯化了截短的蛋白质。我们表明，FixL 从氨基酸残基 127 到残基 260 的片段结合血红素，保留结合氧的能力，并且没有可检测到的激酶活性。 FixL 的 C 端片段从残基 260 开始，无法结合血红素，但具有激酶活性。我们还证明，厌氧导致体外 FixL* 自磷酸化和 FixJ 磷酸化活性增强。最后，我们证明 FixL 的血红素结合区域是体外氧调节其激酶活性所必需的。
Transcription of nitrogen fixation (nif and fix) genes in Rhizobium meliloti is induced by a decrease in oxygen concentration. The products of two genes, fixL and fixJ, are responsible for sensing and transmitting the low-oxygen signal. The proteins encoded by fixL and fixJ (FixL and FixJ, respectively) are homologous to a family of bacterial proteins that transduce environmental signals through a common phosphotransfer mechanism [David, M., Daveran, M., Batut, J., Dedieu, A., Domergue, O., Ghai, J., Hertig, C., Boistard, P. & Khan, D. (1988) Cell 54, 671-683]. FixL, the oxygen sensor, is a membrane protein. It has previously been shown that a soluble derivative of FixL, FixL*, is an oxygen-binding hemoprotein and a kinase that autophosphorylates and also phosphorylates FixJ [Gilles-Gonzalez, M. A., Ditta, G. S. & Helinski, D. R. (1991) Nature (London) 350, 170-172]. In this work, deletion derivatives of fixL* were constructed and overexpressed in Escherichia coli, and the truncated proteins were purified. We show that a fragment of FixL from amino acid residue 127 to residue 260 binds heme, retains the ability to bind oxygen, and has no detectable kinase activity. A C-terminal fragment of FixL, beginning at residue 260, fails to bind heme but is active as a kinase. We also demonstrate that anaerobiosis results in an enhancement of FixL* autophosphorylation and FixJ phosphorylation activities in vitro. Finally, we show that the heme-binding region of FixL is required in vitro for oxygen regulation of its kinase activities.