Role of the dinitrogenase reductase arginine 101 residue in dinitrogenase reductase ADP-ribosyltransferase binding, NAD binding, and cleavage.
Role of the dinitrogenase reductase arginine 101 residue in dinitrogenase reductase ADP-ribosyltransferase binding, NAD binding, and cleavage.
复制标题
二固氮酶还原酶精氨酸 101 残基在二固氮酶还原酶 ADP-核糖基转移酶结合、NAD 结合和裂解中的作用。
DOI：
10.1128/jb.183.1.250-256.2001
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发表时间：
2001
影响因子：
3.2
通讯作者：
Ludden,PW
中科院分区：
文献类型：
--
作者：
Ma,Y;Ludden,PW
在某些细菌中，二硝基酶还原酶通过精氨酸 101 残基的 ADP-核糖基化，受到二硝基酶还原酶 ADP-核糖基转移酶 (DRAT) 的翻译后调节。通过基因。含有R101F形式的二硝基酶还原酶的菌株保留了91％，含有R101Y形式的菌株保留了72％，含有R101L形式的菌株仅保留了含有101位上具有精氨酸的二硝基酶还原酶的菌株的28％的体内固氮酶活性。体内乙炔还原测定，用抗二氮酶还原酶抗体进行免疫印迹，以及[腺苷酸-32P]NAD 标记实验表明，这三个突变体中的任何一个都没有发生固氮酶活性的关闭，并且在体内或体外都没有发生改变的二固氮酶还原酶的 ADP-核糖基化。将来自菌株 UR629 (R101Y) 和 UR630 (R101F) 的改变的二氮酶还原酶纯化至同质。 R101F 和 R101Y 形式的二氮酶还原酶能够与 DRAT 形成复合物，该复合物可以通过 1-乙基-3-(3-二甲基氨基丙基)-碳二亚胺进行化学交联。 R101F 形式的二氮酶还原酶和 DRAT 一起不能裂解 NAD。这表明精氨酸 101 对于 DRAT 与二硝基酶还原酶的结合并不重要，但精氨酸 101 的可用性对于 NAD 裂解很重要。 DRAT 和二氮酶还原酶均可在 UV 照射下被 [羰基-14C]NAD 单独标记，但当两种蛋白质都存在时，大多数 14C 标记会掺入 DRAT 中。 R101F 二硝基酶还原酶被 [羰基-14C]NAD 标记的能力表明 Arg 101 并不是 NAD 结合所绝对必需的。
Dinitrogenase reductase is posttranslationally regulated by dinitrogenase reductase ADP-ribosyltransferase (DRAT) via ADP-ribosylation of the arginine 101 residue in some bacteria.Rhodospirillum rubrumstrains in which the arginine 101 of dinitrogenase reductase was replaced by tyrosine, phenylalanine, or leucine were constructed by site-directed mutagenesis of thenifHgene. The strain containing the R101F form of dinitrogenase reductase retains 91%, the strain containing the R101Y form retains 72%, and the strain containing the R101L form retains only 28% of in vivo nitrogenase activity of the strain containing the dinitrogenase reductase with arginine at position 101. In vivo acetylene reduction assays, immunoblotting with anti-dinitrogenase reductase antibody, and [adenylate-32P]NAD labeling experiments showed that no switch-off of nitrogenase activity occurred in any of the three mutants and no ADP-ribosylation of altered dinitrogenase reductases occurred either in vivo or in vitro. Altered dinitrogenase reductases from strains UR629 (R101Y) and UR630 (R101F) were purified to homogeneity. The R101F and R101Y forms of dinitrogenase reductase were able to form a complex with DRAT that could be chemically cross-linked by 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide. The R101F form of dinitrogenase reductase and DRAT together were not able to cleave NAD. This suggests that arginine 101 is not critical for the binding of DRAT to dinitrogenase reductase but that the availability of arginine 101 is important for NAD cleavage. Both DRAT and dinitrogenase reductase can be labeled by [carbonyl-14C]NAD individually upon UV irradiation, but most14C label is incorporated into DRAT when both proteins are present. The ability of R101F dinitrogenase reductase to be labeled by [carbonyl-14C]NAD suggested that Arg 101 is not absolutely required for NAD binding.