Prudêncio M, Eady R R, Sawers G
Nitrogen Fixation Laboratory, John Innes Centre, Norwich, United Kingdom.
J Bacteriol. 1999 Apr;181(8):2323-9. doi: 10.1128/JB.181.8.2323-2329.1999.
The nirA gene encoding the blue dissimilatory nitrite reductase from Alcaligenes xylosoxidans has been cloned and sequenced. To our knowledge, this is the first report of the characterization of a gene encoding a blue copper-containing nitrite reductase. The deduced amino acid sequence exhibits a high degree of similarity to other copper-containing nitrite reductases from various bacterial sources. The full-length protein included a 24-amino-acid leader peptide. The nirA gene was overexpressed in Escherichia coli and was shown to be exported to the periplasm. Purification was achieved in a single step, and analysis of the recombinant Nir enzyme revealed that cleavage of the signal peptide occurred at a position identical to that for the native enzyme isolated from A. xylosoxidans. The recombinant Nir isolated directly was blue and trimeric and, on the basis of electron paramagnetic resonance spectroscopy and metal analysis, possessed only type 1 copper centers. This type 2-depleted enzyme preparation also had a low nitrite reductase enzyme activity. Incubation of the periplasmic fraction with copper sulfate prior to purification resulted in the isolation of an enzyme with a full complement of type 1 and type 2 copper centers and a high specific activity. The kinetic properties of the recombinant enzyme were indistinguishable from those of the native nitrite reductase isolated from A. xylosoxidans. This rapid isolation procedure will greatly facilitate genetic and biochemical characterization of both wild-type and mutant derivatives of this protein.
编码木糖氧化产碱杆菌蓝色异化型亚硝酸还原酶的nirA基因已被克隆并测序。据我们所知，这是关于编码含蓝色铜的亚硝酸还原酶基因特性的首次报道。推导的氨基酸序列与来自各种细菌来源的其他含铜亚硝酸还原酶具有高度相似性。全长蛋白质包含一个24个氨基酸的前导肽。nirA基因在大肠杆菌中过表达，并被证明分泌到周质中。一步实现了纯化，重组Nir酶的分析表明信号肽的切割发生在与从木糖氧化产碱杆菌分离的天然酶相同的位置。直接分离的重组Nir呈蓝色且为三聚体，基于电子顺磁共振光谱和金属分析，仅具有1型铜中心。这种不含2型铜的酶制剂也具有低亚硝酸还原酶活性。在纯化前将周质部分与硫酸铜一起孵育，导致分离出一种具有完整1型和2型铜中心且比活性高的酶。重组酶的动力学特性与从木糖氧化产碱杆菌分离的天然亚硝酸还原酶的动力学特性无法区分。这种快速分离程序将极大地促进该蛋白质野生型和突变体衍生物的遗传和生化特性研究。
