Harris G S, White T C, Flory J E, Orme-Johnson W H
Department of Chemistry, Massachusetts Institute of Technology, Cambridge 02139.
J Biol Chem. 1990 Sep 15;265(26):15909-19.
A binary plasmid system was used to produce nitrogenase components in Escherichia coli and subsequently to define a minimum set of nitrogen fixation (nif) genes required for the production of the iron-molybdenum cofactor (FeMoco) reactivatable apomolybdenum-iron (apoMoFe) protein of nitrogenase. The active MoFe protein is an alpha 2 beta 2 tetramer containing two FeMoco clusters and 4 Fe4S4 P centers (for review see, Orme-Johnson, W.H. (1985) Annu. Rev. Biophys. Biophys. Chem. 14, 419-459). The plasmid pVL15, carrying a tac-promoted nifA activator gene, was coharbored in E. coli with the plasmid pGH1 which contained nifHDKTYENXUSVWZMF' derived from the chromosome of the nitrogen fixing bacterium Klebsiella pneumoniae. The apoMoFe protein produced in E. coli by pGH1 + VL15 was identical to the apoprotein in derepressed cells of the nifB- mutant of K. pneumoniae (UN106) in its electrophoretic properties on nondenaturing polyacrylamide gels as well as in its ability to be activated by FeMoco. The constituent peptides migrated identically to those from purified MoFe protein during electrophoresis on denaturing gels. The concentrations of apoMoFe protein produced in nif-transformed strains of E. coli were greater than 50% of the levels of MoFe protein observed in derepressed wild-type K. pneumoniae. Systematic deletion of individual nif genes carried by pGH1 has established the requirements for the maximal production of the FeMoco-reactivatable apoMoFe protein to be the following gene products, NifHDKTYUSWZM+A. It appears that several of the genes (nifT, Y, U, W, and Z) are only required for maximal production of the apoMoFe protein, while others (nifH, D, K, and S) are absolutely required for synthesis of this protein in E. coli. One curious result is that the nifH gene product, the peptide of the Fe protein, but not active Fe protein itself, is required for formation of the apoMoFe protein. This suggests the possibility of a ternary complex of the NifH, D, and K peptides as the substrate for the processing to form the apoMoFe protein. We also find that nifM, the gene which processes the nifH protein into Fe protein (Howard, K.S., McLean, P.A., Hansen, F. B., Lemley, P.V., Kobla, K.S. & Orme-Johnson, W.H. (1986) J. Biol. Chem. 261, 772-778) can, under certain circumstances, partially replace other processing genes (i.e. nifTYU and/or WZ) although it is not essential for apoMoFe protein formation. It also appears that nifS and nifU, reported to play a role in Fe protein production in Azotobacter vinelandii, play no such role in K. pneumoniae, although these genes are involved in apoMoFe formation.(ABSTRACT TRUNCATED AT 400 WORDS)
采用二元质粒系统在大肠杆菌中生产固氮酶组分，进而确定生产可被铁钼辅因子（FeMoco）激活的固氮酶脱辅钼铁（apoMoFe）蛋白所需的最小固氮（nif）基因集。活性钼铁蛋白是一种α2β2四聚体，包含两个FeMoco簇和4个Fe4S4 P中心（综述见Orme-Johnson, W.H. (1985) Annu. Rev. Biophys. Biophys. Chem. 14, 419 - 459）。携带tac启动子驱动的nifA激活基因的质粒pVL15，与含有源自固氮细菌肺炎克雷伯氏菌染色体的nifHDKTYENXUSVWZMF'的质粒pGH1共存在大肠杆菌中。pGH1 + VL15在大肠杆菌中产生的apoMoFe蛋白，在非变性聚丙烯酰胺凝胶上的电泳性质以及被FeMoco激活的能力方面，与肺炎克雷伯氏菌nifB - 突变体（UN106）去阻遏细胞中的脱辅基蛋白相同。在变性凝胶上电泳时，其组成肽段与纯化的钼铁蛋白的肽段迁移情况相同。在经nif转化的大肠杆菌菌株中产生的apoMoFe蛋白浓度，大于去阻遏的野生型肺炎克雷伯氏菌中观察到的钼铁蛋白水平的50%。对pGH1携带的各个nif基因进行系统缺失，已确定FeMoco可激活的apoMoFe蛋白最大产量所需的基因产物如下：NifHDKTYUSWZM + A。似乎其中几个基因（nifT、Y、U、W和Z）仅对apoMoFe蛋白的最大产量是必需的，而其他基因（nifH、D、K和S）对于在大肠杆菌中合成该蛋白是绝对必需的。一个奇怪的结果是，固氮酶铁蛋白的肽段nifH基因产物，但不是活性铁蛋白本身，是形成apoMoFe蛋白所必需的。这表明存在NifH、D和K肽段的三元复合物作为加工形成apoMoFe蛋白的底物的可能性。我们还发现，将nifH蛋白加工成铁蛋白的基因nifM（Howard, K.S., McLean, P.A., Hansen, F. B., Lemley, P.V., Kobla, K.S. & Orme-Johnson, W.H. (1986) J. Biol. Chem. 261, 772 - 778）在某些情况下可以部分替代其他加工基因（即nifTYU和/或WZ），尽管它对于apoMoFe蛋白的形成不是必需的。似乎据报道在棕色固氮菌铁蛋白生产中起作用的nifS和nifU，在肺炎克雷伯氏菌中不起这样的作用，尽管这些基因参与apoMoFe蛋白的形成。（摘要截短于4**00字）
