Journal
BIOCHIMICA ET BIOPHYSICA ACTA-BIOENERGETICS
Volume 1837, Issue 7, Pages 1122-1129Publisher
ELSEVIER
DOI: 10.1016/j.bbabio.2013.12.006
Keywords
Klebsiella pneumoniae; NADH:quinone oxidoreductase; Fumarate reductase; Flavin transferase; Na+ transport; FMN
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Funding
- Russian Foundation for Basic Research [13-04-00332, 12-04-01002]
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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 99.5 kDa 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 1 mol/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. (C) 2013 Elsevier B.V. All rights reserved.
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