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

FEBS Letters

Finel, Moshe¹ 

[1] Helsinki Bioenergetics Group, Department of Medical Chemistry, Institute of Biomedicine, University of Helsinki, P. O. Box 8 (Siltvuorenpenger 10), FIN-00140 Helsinki, Finland

关键词: NDH1;    NDH2;    Complex I;    Respiratory chain;    Membrane protein;    NDH;    NADH;    quinone oxidoreductase (NADH dehydrogenase);    MDH;    (S)-Mandelate dehydrogenase;   

DOI  :  10.1016/0014-5793(96)00831-9

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 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.

【 授权许可】

Unknown