Arese Marzia, Zumft Walter G, Cutruzzolà Francesca
Department of Biochemical Sciences, University of Rome La Sapienza, P.le A.Moro 5, 00185 Rome, Italy.
Protein Expr Purif. 2003 Jan;27(1):42-8. doi: 10.1016/s1046-5928(02)00600-9.
Nitrite reductases are redox enzymes catalysing the one electron reduction of nitrite to nitrogen monoxide (NO) within the bacterial denitrification process. We have cloned the gene for cd(1) nitrite reductase (Pa-nirS) from Pseudomonas aeruginosa into the NiRS(-) strain MK202 of Pseudomonas stutzeri and expressed the enzyme under denitrifying conditions. In the MK202 strain, denitrification is abolished by the disruption of the endogenous nitrite reductase gene; thus, cells can be grown only in the presence of oxygen. After complementation with Pa-nirS gene, cells supplemented with nitrate can be grown in the absence of oxygen. The presence of nitrite reductase was proven in vivo by the demonstration of NO production, showing that the enzyme was expressed in the active form, containing both heme c and d(1). A purification procedure for the recombinant PaNir has been developed, based on the P. aeruginosa purification protocol; spectroscopic analysis of the purified protein fully confirms the presence of the d(1) heme cofactor. Moreover, the functional characterisation of the recombinant NiR has been carried out by monitoring the production of NO by the purified NiR enzyme in the presence of nitrite by an NO electrode. The full recovery of the denitrification properties in the P. stutzeri MK202 strain by genetic complementation with Pa-NiR underlines the high homology between enzymes of nitrogen oxianion respiration. Our work provides an expression system for cd(1) nitrite reductase and its site-directed mutants in a non-pathogenic strain and is a starting point for the in vivo study of recombinant enzyme variants.
亚硝酸还原酶是一类氧化还原酶，在细菌反硝化过程中催化亚硝酸单电子还原为一氧化氮（NO）。我们已将铜绿假单胞菌的cd(1)亚硝酸还原酶基因（Pa-nirS）克隆到施氏假单胞菌的NiRS(-)菌株MK202中，并在反硝化条件下表达该酶。在MK202菌株中，内源性亚硝酸还原酶基因的破坏消除了反硝化作用；因此，细胞只能在有氧条件下生长。用Pa-nirS基因互补后，添加硝酸盐的细胞可以在无氧条件下生长。通过证明NO的产生在体内证实了亚硝酸还原酶的存在，表明该酶以活性形式表达，同时含有血红素c和d(1)。基于铜绿假单胞菌的纯化方案，开发了一种重组PaNir的纯化程序；对纯化蛋白的光谱分析充分证实了d(1)血红素辅因子的存在。此外，通过用NO电极监测纯化的NiR酶在亚硝酸盐存在下NO的产生，对重组NiR进行了功能表征。通过用Pa-NiR进行基因互补，施氏假单胞菌MK202菌株的反硝化特性完全恢复，这突出了氮氧阴离子呼吸酶之间的高度同源性。我们的工作为非致病菌株中的cd(1)亚硝酸还原酶及其定点突变体提供了一个表达系统，是重组酶变体体内研究的起点。
