Tungstate Uptake by a highly specific ABC transporter in Eubacterium acidaminophilum.
The Gram-positive anaerobe Eubacterium acidaminophilum contains at least two tungsten-dependent enzymes: viologen-dependent formate dehydrogenase and aldehyde dehydrogenase. (185)W-Labeled tungstate was taken up by this organism with a maximum rate of 0.53 pmol min(-)1 mg(-)1 of protein at 36 degrees C. The uptake was not affected by equimolar amounts of molybdate. The genes tupABC coding for an ABC transporter specific for tungstate were cloned in the downstream region of genes encoding a tungsten-containing formate dehydrogenase. The substrate-binding protein, TupA, of this putative transporter was overexpressed in Escherichia coli, and its binding properties toward oxyanions were determined by a native polyacrylamide gel retardation assay. Only tungstate induced a shift of TupA mobility, suggesting that only this anion was specifically bound by TupA. If molybdate and sulfate were added in high molar excess (>1000-fold), they were also slightly bound by TupA. The K(d) value for tungstate was determined to be 0.5 microm. The genes encoding the tungstate-specific ABC transporter exhibited highest similarities to putative transporters from Methanobacterium thermoautotrophicum, Haloferax volcanii, Vibrio cholerae, and Campylobacter jejuni. These five transporters represent a separate phylogenetic group of oxyanion ABC transporters as evident from analysis of the deduced amino acid sequences of the binding proteins. Downstream of the tupABC genes, the genes moeA, moeA-1, moaA, and a truncated moaC have been identified by sequence comparison of the deduced amino acid sequences. They should participate in the biosynthesis of the pterin cofactor that is present in molybdenum- and tungsten-containing enzymes except nitrogenase.
Makdessi
K
K
Institut für Mikrobiologie, Martin-Luther-Universität Halle-Wittenberg, Kurt-Mothes-Strasse 3, 06120 Halle, Germany.
Andreesen
J R
JR
Pich
A
A
D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000444:Aldehyde Dehydrogenase / Q000378:metabolism; D000595:Amino Acid Sequence; D000838:Anions / Q000378:metabolism; D001426:Bacterial Proteins; D003001:Cloning, Molecular; D005051:Eubacterium / Q000378:metabolism*; D005560:Formate Dehydrogenases / Q000378:metabolism; D007700:Kinetics; D008291:Malate Dehydrogenase / Q000378:metabolism; D008969:Molecular Sequence Data; D008982:Molybdenum / Q000378:metabolism; D009876:Operon; D011487:Protein Conformation; D016415:Sequence Alignment; D013431:Sulfates / Q000378:metabolism; D017973:Tungsten Compounds / Q000493:pharmacokinetics*; D014755:Viologens / Q000378:metabolism
D018528:ATP-Binding Cassette Transporters; D000838:Anions; D001426:Bacterial Proteins; D013431:Sulfates; D017973:Tungsten Compounds; C431931:TupA protein, Eubacterium acidaminophilum; C431932:TupB protein, Eubacterium acidaminophilum; C431933:TupC protein, Eubacterium acidaminophilum; D014755:Viologens; C044659:molybdate; D008982:Molybdenum; D008291:Malate Dehydrogenase; C031917:malate dehydrogenase (oxaloacetate-decarboxylating) (NADP+); D005560:Formate Dehydrogenases; D000444:Aldehyde Dehydrogenase; C045951:tungstate