Hutchings M I, Shearer N, Wastell S, van Spanning R J, Spiro S
School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, United Kingdom.
J Bacteriol. 2000 Nov;182(22):6434-9. doi: 10.1128/JB.182.22.6434-6439.2000.
The transcription factor NNR from Paracoccus denitrificans was expressed in a strain of Escherichia coli carrying a plasmid-borne fusion of the melR promoter to lacZ, with a consensus FNR-binding site 41.5 bp upstream of the transcription start site. This promoter was activated by NNR under anaerobic growth conditions in media containing nitrate, nitrite, or the NO(+) donor sodium nitroprusside. Activation by nitrate was abolished by a mutation in the molybdenum cofactor biosynthesis pathway, indicating a requirement for nitrate reductase activity. Activation by nitrate was modulated by the inclusion of reduced hemoglobin in culture media, because of the ability of hemoglobin to sequester nitric oxide and nitrite. The ability of nitrate and nitrite to activate NNR is likely due to the formation of NO (or related species) during nitrate and nitrite respiration. Amino acids potentially involved in NNR activity were replaced by site-directed mutagenesis, and the activities of NNR derivatives were tested in the E. coli reporter system. Substitutions at Cys-103 and Tyr-35 significantly reduced NNR activity but did not abolish the response to reactive nitrogen species. Substitutions at Phe-82 and Tyr-93 severely impaired NNR activity, but the altered proteins retained the ability to repress an FNR-repressible promoter, so these mutations have a "positive control" phenotype. It is suggested that Phe-82 and Tyr-93 identify an activating region of NNR that is involved in an interaction with RNA polymerase. Replacement of Ser-96 with alanine abolished NNR activity, and the protein was undetectable in cell extracts. In contrast, NNR in which Ser-96 was replaced with threonine retained full activity.
反硝化副球菌的转录因子NNR在携带melR启动子与lacZ的质粒融合体的大肠杆菌菌株中表达，转录起始位点上游41.5 bp处有一个共有FNR结合位点。在含有硝酸盐、亚硝酸盐或NO(+)供体硝普钠的培养基中，厌氧生长条件下NNR可激活该启动子。钼辅因子生物合成途径中的突变消除了硝酸盐的激活作用，表明需要硝酸还原酶活性。由于血红蛋白能够螯合一氧化氮和亚硝酸盐，因此培养基中加入还原血红蛋白可调节硝酸盐的激活作用。硝酸盐和亚硝酸盐激活NNR的能力可能是由于在硝酸盐和亚硝酸盐呼吸过程中形成了NO（或相关物质）。通过定点诱变替换了可能参与NNR活性的氨基酸，并在大肠杆菌报告系统中测试了NNR衍生物的活性。Cys-103和Tyr-35处的替换显著降低了NNR活性，但并未消除对活性氮物质的反应。Phe-82和Tyr-93处的替换严重损害了NNR活性，但改变后的蛋白质仍保留了抑制FNR可抑制启动子的能力，因此这些突变具有“正调控”表型。有人认为，Phe-82和Tyr-93确定了NNR的一个激活区域，该区域参与与RNA聚合酶的相互作用。用丙氨酸替换Ser-96消除了NNR活性，并且在细胞提取物中检测不到该蛋白质。相反，用苏氨酸替换Ser-96的NNR保留了全部活性。
