Pulakat L, Hausman B S, Lei S, Gavini N
Department of Biological Sciences, Bowling Green State University, Ohio 43403, USA.
J Biol Chem. 1996 Jan 26;271(4):1884-9. doi: 10.1074/jbc.271.4.1884.
We have identified the molecular basis for the nitrogenase negative phenotype exhibited by Azotobacter vinelandii UW97. This strain was initially isolated following nitrosoguanidine mutagenesis. Recently, it was shown that this strain lacks the Fe protein activity, which results in the synthesis of a FeMo cofactor-deficient apodinitrogenase. Activation of this apodinitrogenase requires the addition of both MgATP and wild-type Fe protein to the crude extracts made by A. vinelandii UW97 (Allen, R.M., Homer, M.J., Chatterjee R., Ludden, P.W., Roberts, G.P., and Shah, V.K. (1993) J. Biol. Chem. 268 23670-23674). Earlier, we proposed the sequence of events in the MoFe protein assembly based on the biochemical and spectroscopic analysis of the purified apodinitrogenase from A. vinelandii DJ54 (Gavini, N., Ma, L., Watt, G., and Burgess, B.K. (1994) Biochemistry 33, 11842-11849). Taken together, these results imply that the assembly process of apodinitrogenase is arrested at the same step in both of these strains. Since A. vinelandii DJ54 is a delta nifH strain, this strain is not useful in identifying the features of the Fe protein involved in the MoFe protein assembly. Here, we report a systematic analysis of an A. vinelandii UW97 mutant and show that, unlike A. vinelandii DJ54, the nifH gene of A. vinelandii UW97 has no deletion in either coding sequence or the surrounding sequences. The specific mutation responsible for the Nif- phenotype of A. vinelandii UW97 is the substitution of a non-conserved serine at position 44 of the Fe protein by a phenylalanine as shown by DNA sequencing. Furthermore, oligonucleotide site-directed mutagenesis was employed to confirm that the Nif- phenotype in A. vinelandii UW97 is exclusively due to the substitution of the Fe protein residue serine 44 by phenylalanine. By contrast, replacing Ser-44 with alanine did not affect the Nif phenotype of A. vinelandii. Therefore, it seems that the Nif- phenotype of A. vinelandii UW97 is caused by a general structural disturbance of the Fe protein due to the presence of the bulky phenylalanine at position 44.
我们已经确定了维涅兰德固氮菌UW97所表现出的固氮酶阴性表型的分子基础。该菌株最初是在亚硝基胍诱变后分离得到的。最近研究表明，该菌株缺乏铁蛋白活性，这导致合成了一种缺乏铁钼辅因子的脱辅基固氮酶。要激活这种脱辅基固氮酶，需要向维涅兰德固氮菌UW97制备的粗提物中添加MgATP和野生型铁蛋白（艾伦，R.M.，霍默，M.J.，查特吉，R.，卢登，P.W.，罗伯茨，G.P.，以及沙阿，V.K.（1993年）《生物化学杂志》268卷，23670 - 23674页）。此前，我们基于对维涅兰德固氮菌DJ54纯化的脱辅基固氮酶的生化和光谱分析，提出了钼铁蛋白组装过程中的事件序列（加维尼，N.，马，L.，瓦特，G.，以及伯吉斯，B.K.（1994年）《生物化学》33卷，11842 - 11849页）。综合这些结果表明，在这两种菌株中，脱辅基固氮酶的组装过程都在同一步骤受阻。由于维涅兰德固氮菌DJ54是一种nifH缺失菌株，该菌株对于鉴定参与钼铁蛋白组装的铁蛋白特征并无帮助。在此，我们报告了对维涅兰德固氮菌UW97一个突变体的系统分析，并表明，与维涅兰德固氮菌DJ54不同，维涅兰德固氮菌UW97的nifH基因在编码序列或其周围序列中均无缺失。如DNA测序所示，导致维涅兰德固氮菌UW97固氮阴性表型的特定突变是铁蛋白第44位的一个非保守丝氨酸被苯丙氨酸取代。此外，采用寡核苷酸定点诱变来确认维涅兰德固氮菌UW97中的固氮阴性表型完全是由于铁蛋白残基丝氨酸44被苯丙氨酸取代所致。相比之下，将丝氨酸 - 44替换为丙氨酸并不影响维涅兰德固氮菌的固氮表型。因此，维涅兰德固氮菌UW97的固氮阴性表型似乎是由于第44位存在庞大的苯丙氨酸导致铁蛋白的整体结构紊乱所致。
