Finel M
Department of Medical Chemistry, University of Helsinki, Finland.
FEBS Lett. 1996 Sep 9;393(1):81-5. doi: 10.1016/0014-5793(96)00831-9.
The H(+)-translocating NADH:ubiquinone oxidoreductase (NDH1) is probably an obligatory enzyme in Paracoccus denitrificans and disruption of its genes may be lethal to this organism. In order to overcome this problem and delete the nqo8 and nqo9 genes of NDH1, it was necessary to render the enzyme non-essential. This was achieved by constructing a deletion plasmid in which most of the coding regions of nqo8 and nqo9 were replaced by the ndh gene of Escherichia coli that encodes an alternative NADH:ubiquinone oxidoreductase (NDH2), and a kanamycin resistance gene. Subsequent homologous recombination gave rise to a mutant the membranes of which catalyzed rotenone-insensitive NADH oxidation, but which did not oxidize deamino-NADH. Hence, this mutant expressed active and membrane-bound NDH2, and lacked NDH1 activity.
H(+)转运型NADH：泛醌氧化还原酶（NDH1）可能是反硝化副球菌中的一种必需酶，其基因的破坏可能对该生物体是致命的。为了克服这个问题并删除NDH1的nqo8和nqo9基因，有必要使该酶变得非必需。这是通过构建一个缺失质粒来实现的，其中nqo8和nqo9的大部分编码区域被大肠杆菌的ndh基因取代，该基因编码另一种NADH：泛醌氧化还原酶（NDH2）以及一个卡那霉素抗性基因。随后的同源重组产生了一个突变体，其膜催化对鱼藤酮不敏感的NADH氧化，但不氧化脱氨基-NADH。因此，这个突变体表达了有活性的膜结合型NDH2，并且缺乏NDH1活性。
