title: Localization-controlled specificity of FAD:threonine flavin transferases in Klebsiella pneumoniae and its implications for the mechanism of Na(+)-translocating NADH:quinone oxidoreductase.
abstract: 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.
authors.affiliation: Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow 119992, Russia.
authors.forename: Yulia V
authors.initials: YV
authors.lastname: Bertsova
authors.affiliation: Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow 119992, Russia.
authors.forename: Vitaly A
authors.initials: VA
authors.lastname: Kostyrko
authors.affiliation: Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow 119992, Russia.
authors.forename: Alexander A
authors.initials: AA
authors.lastname: Baykov
authors.affiliation: Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow 119992, Russia. Electronic address: bogachev@belozersky.msu.ru.
authors.forename: Alexander V
authors.initials: AV
authors.lastname: Bogachev
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D020134:Catalytic Domain; D003593:Cytoplasm / Q000378:metabolism; D005415:Flavins / Q000378:metabolism; D007711:Klebsiella pneumoniae / Q000201:enzymology* / Q000378:metabolism; D008969:Molecular Sequence Data; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011808:Quinone Reductases / Q000737:chemistry / Q000378:metabolism*; D012964:Sodium / Q000378:metabolism; D013379:Substrate Specificity; D013385:Succinate Dehydrogenase / Q000737:chemistry / Q000378:metabolism; D013912:Threonine / Q000378:metabolism; D014734:Vibrio cholerae / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D005415:Flavins; D013912:Threonine; D012964:Sodium; D013385:Succinate Dehydrogenase; D009247:NADH, NADPH Oxidoreductases; C084142:sodium-translocating NADH-quinone reductase; D011808:Quinone Reductases
keywords: FMN; Flavin transferase; Fumarate reductase; Klebsiella pneumoniae; NADH:quinone oxidoreductase; Na(+) transport