Curdt I, Singh B B, Jakoby M, Hachtel W, Böhme H
Botanisches Institut der Universität Bonn, Karlrobert-Kreiten-Strasse 13, D-53115 Bonn, Germany.
Biochim Biophys Acta. 2000 Nov 30;1543(1):60-8. doi: 10.1016/s0167-4838(00)00198-9.
The nitrite reductase gene (nirA) from the filamentous, heterocyst-forming cyanobacterium Anabaena sp. PCC 7120 (A. PCC 7120) was expressed in Escherichia coli using the pET-system. Co-expression of the cysG gene encoding siroheme synthase of Salmonella typhimurium increased the amount of soluble, active nitrite reductase four fold. Nitrite reductase was purified to homogeneity. In order to identify amino acid residues involved in ferredoxin (PetF)-nitrite reductase electron transfer in A. PCC 7120, we performed a sequence comparison between ferredoxin-dependent nitrite reductases from various species. The alignment revealed a number of conserved residues possibly involved in ferredoxin nitrite reductase interaction. The position of these residues relative to the [4Fe4S]-cluster as the primary electron acceptor was tentatively localized in a three dimensional structure of the sulfite reductase from E. coli, which is closest related to nitrite reductase among the proteins with known tertiary structure. The exchange of certain positively charged amino acid residues of the nitrite reductase with uncharged residues revealed the influence of these residues on the interaction of nitrite reductase with reduced ferredoxin. We identified at least two separate regions of nitrite reductase that contribute to the binding of ferredoxin.
利用pET系统在大肠杆菌中表达了丝状、异形胞形成的蓝藻鱼腥藻Anabaena sp. PCC 7120（A. PCC 7120）的亚硝酸还原酶基因（nirA）。共表达编码鼠伤寒沙门氏菌的西罗血红素合酶的cysG基因，可使可溶性活性亚硝酸还原酶的量增加四倍。亚硝酸还原酶被纯化至同质。为了鉴定参与A. PCC 7120中铁氧还蛋白（PetF）-亚硝酸还原酶电子转移的氨基酸残基，我们对来自不同物种的铁氧还蛋白依赖性亚硝酸还原酶进行了序列比较。比对结果显示了一些可能参与铁氧还蛋白与亚硝酸还原酶相互作用的保守残基。这些残基相对于作为主要电子受体的[4Fe4S]簇的位置，暂定位在大肠杆菌亚硫酸盐还原酶的三维结构中，在已知三级结构的蛋白质中，该亚硫酸盐还原酶与亚硝酸还原酶关系最为密切。将亚硝酸还原酶的某些带正电荷的氨基酸残基替换为不带电荷的残基，揭示了这些残基对亚硝酸还原酶与还原型铁氧还蛋白相互作用的影响。我们确定了亚硝酸还原酶中至少两个独立的区域，它们有助于铁氧还蛋白的结合。
