Genetic inactivation of the H+-translocating NADH:ubiquinone oxidoreductase of Paracoccus denitrificans is facilitated by insertion of the ndh gene from Escherichia coli

被引:24
作者
Finel, M
机构
[1] Helsinki Bioenergetics Group, Department of Medical Chemistry, University of Helsinki, FIN-00140 Helsinki
基金
芬兰科学院;
关键词
NADH1; NADH2; complex I; respiratory chain; membrane protein;
D O I
10.1016/0014-5793(96)00831-9
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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.
引用
收藏
页码:81 / 85
页数:5
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