Glockner A B, Jüngst A, Zumft W G
Lehrstuhl für Mikrobiologie, Universität Karlsruhe, Germany.
Arch Microbiol. 1993;160(1):18-26. doi: 10.1007/BF00258141.
The structural gene, nirK, for the respiratory Cu-containing nitrite reductase from denitrifying Pseudomonas aureofaciens was isolated and sequenced. It encodes a polypeptide of 363 amino acids including a signal peptide of 24 amino acids for protein export. The sequence showed 63.8% positional identity with the amino acid sequence of "Achromobacter cycloclastes" nitrite reductase. Ligands for the blue, type I Cu-binding site and for a putative type-II site were identified. The nirK gene was transferred to the mutant MK202 of P. stutzeri which lacks cytochrome cd1 nitrite reductase due to a transposon Tn5 insertion in its structural gene, nirS. The heterologous enzyme was active in vitro and in vivo in this background and restored the mutationally interrupted denitrification pathway. Transfer of nirK to Escherichia coli resulted in an active nitrite reductase in vitro. Expression of the nirS gene from P. stutzeri in P. aureofaciens and E. coli led to nonfunctional gene products. Nitrite reductase activity of cell extract from either bacterium could be reconstituted by addition of heme d1, indicating that both heterologous hosts synthesized a cytochrome cd1 without the d1-group.
从反硝化的金黄色假单胞菌中分离并测序了编码含铜呼吸型亚硝酸还原酶的结构基因nirK。它编码一个由363个氨基酸组成的多肽，其中包括一个用于蛋白质输出的24个氨基酸的信号肽。该序列与“环裂无色杆菌”亚硝酸还原酶的氨基酸序列在位置上有63.8%的一致性。确定了蓝色I型铜结合位点和一个假定的II型位点的配体。nirK基因被转移到施氏假单胞菌的突变体MK202中，该突变体由于其结构基因nirS中插入转座子Tn5而缺乏细胞色素cd1亚硝酸还原酶。在这种背景下，异源酶在体外和体内均具有活性，并恢复了因突变而中断的反硝化途径。将nirK转移到大肠杆菌中，在体外产生了一种有活性的亚硝酸还原酶。施氏假单胞菌的nirS基因在金黄色假单胞菌和大肠杆菌中的表达产生了无功能的基因产物。通过添加血红素d1可以重建来自任何一种细菌的细胞提取物的亚硝酸还原酶活性，这表明两种异源宿主都合成了不含d1基团的细胞色素cd1。
