Bertsova Yulia V, Kostyrko Vitaly A, Baykov Alexander A, Bogachev Alexander V
Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow 119992, Russia.
Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow 119992, Russia.
Biochim Biophys Acta. 2014 Jul;1837(7):1122-9. doi: 10.1016/j.bbabio.2013.12.006. Epub 2013 Dec 20.
The Klebsiella pneumoniae genome contains genes for two putative flavin transferase enzymes (ApbE1 and ApbE2) that add FMN to protein Thr residues. ApbE1, but not ApbE2, has a periplasm-addressing signal sequence. The genome also contains genes for three target proteins with the Dxx(s/t)gAT flavinylation motif: two subunits of Na(+)-translocating NADH:quinone oxidoreductase (Na(+)-NQR), and a 99.5kDa protein, KPK_2907, with a previously unknown function. We show here that KPK_2907 is an active cytoplasmically-localized fumarate reductase. K. pneumoniae cells with an inactivated kpk_2907 gene lack cytoplasmic fumarate reductase activity, while retaining this activity in the membrane fraction. Complementation of the mutant strain with a kpk_2907-containing plasmid resulted in a complete recovery of cytoplasmic fumarate reductase activity. KPK_2907 produced in Escherichia coli cells contains 1mol/mol each of covalently bound FMN, noncovalently bound FMN and noncovalently bound FAD. Lesion in the ApbE1 gene in K. pneumoniae resulted in inactive Na(+)-NQR, but cytoplasmic fumarate reductase activity remained unchanged. On the contrary, lesion in the ApbE2 gene abolished the fumarate reductase but not the Na(+)-NQR activity. Both activities could be restored by transformation of the ApbE1- or ApbE2-deficient K. pneumoniae strains with plasmids containing the Vibrio cholerae apbE gene with or without the periplasm-directing signal sequence, respectively. Our data thus indicate that ApbE1 and ApbE2 bind FMN to Na(+)-NQR and fumarate reductase, respectively, and that, contrary to the presently accepted view, the FMN residues are on the periplasmic side of Na(+)-NQR. A new, "electron loop" mechanism is proposed for Na(+)-NQR, involving an electroneutral Na(+)/electron symport. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference.
肺炎克雷伯菌基因组包含两个假定的黄素转移酶（ApbE1和ApbE2）基因，它们将FMN添加到蛋白质苏氨酸残基上。ApbE1具有周质定位信号序列，而ApbE2没有。该基因组还包含三个具有Dxx(s/t)gAT黄素化基序的靶蛋白基因：Na(+)-转运NADH：醌氧化还原酶（Na(+)-NQR）的两个亚基，以及一个功能未知的99.5 kDa蛋白KPK_2907。我们在此表明，KPK_2907是一种活跃的定位于细胞质的延胡索酸还原酶。kpk_2907基因失活的肺炎克雷伯菌细胞缺乏细胞质延胡索酸还原酶活性，但膜部分保留了这种活性。用含kpk_2907的质粒对突变菌株进行互补，可使细胞质延胡索酸还原酶活性完全恢复。在大肠杆菌细胞中产生的KPK_2907，每摩尔含有1摩尔共价结合的FMN、非共价结合的FMN和非共价结合的FAD。肺炎克雷伯菌ApbE1基因的损伤导致Na(+)-NQR失活，但细胞质延胡索酸还原酶活性保持不变。相反，ApbE2基因的损伤消除了延胡索酸还原酶活性，但不影响Na(+)-NQR活性。分别用含有霍乱弧菌apbE基因（有或无周质导向信号序列）的质粒转化ApbE1或ApbE2缺陷的肺炎克雷伯菌菌株，可恢复这两种活性。因此，我们的数据表明，ApbE1和ApbE2分别将FMN与Na(+)-NQR和延胡索酸还原酶结合，并且与目前公认的观点相反，FMN残基位于Na(+)-NQR的周质侧。我们提出了一种新的“电子循环”机制用于Na(+)-NQR，涉及电中性的Na(+)/电子同向转运。本文是名为：第18届欧洲生物能量学会议的特刊的一部分。
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