RESUMO
The genome of the Honeybee bacterial pathogen, Paenibacillus larvae, encodes for protein a with substantial amino acid sequence similarity to the canonical Escherichia coli uracil transporter UraA. P. larvae expresses the uracil permease (PlUP) locus, and is sensitive to the presence of the toxic uracil analog 5-fluorouracil under vegetative growth conditions. The solute transport and binding profile of PlUP was determined by radiolabeled uptake experiments via heterologous expression in nucleobase transporter-deficient Saccharomyces cerevisiae strains. PlUP is specific for the transport of uracil and competitively binds xanthine and uric acid. Further biochemical characterization reveals that PlUP has a strong affinity for uracil with a Km 19.5⯱â¯1.6⯵M. Uracil transport is diminished in the presence of the proton disruptor carbonyl cyanide m-chlorophenylhydrazone, but not by the sodium gradient disruptor Ouabain.
Assuntos
Proteínas de Bactérias/metabolismo , Abelhas/microbiologia , Proteínas de Membrana Transportadoras/metabolismo , Paenibacillus larvae/metabolismo , Uracila/metabolismo , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Transporte Biológico , Cinética , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/genética , Paenibacillus larvae/química , Paenibacillus larvae/genética , Especificidade por Substrato , Uracila/química , Ácido Úrico/química , Ácido Úrico/metabolismo , Xantina/química , Xantina/metabolismoRESUMO
Here, we report that a novel nucleobase cation symporter 2 encoded in the genome of the honeybee bacterial pathogen Paenibacillus larvae reveals high levels of amino acid sequence similarity to the Escherichia coli and Bacillus subtilis uric acid and xanthine transporters. This transporter is named P. larvae uric acid permease-like protein (PlUacP). Even though PlUacP displays overall amino acid sequence similarities, has common secondary structures, and shares functional motifs and functionally important amino acids with E. coli xanthine and uric acid transporters, these commonalities are insufficient to assign transport function to PlUacP. The solute transport and binding profile of PlUacP was determined by radiolabeled uptake experiments via heterologous expression in nucleobase transporter-deficient Saccharomyces cerevisiae strains. PlUacP transports the purines adenine and guanine and the pyrimidine uracil. Hypoxanthine, xanthine, and cytosine are not transported by PlUacP, but, along with uric acid, bind in a competitive manner. PlUacP has strong affinity for adenine Km 7.04 ± 0.18 µm, and as with other bacterial and plant NCS2 proteins, PlUacP function is inhibited by the proton disruptor carbonyl cyanide m-chlorophenylhydrazone. The solute transport and binding profile identifies PlUacP as a novel nucleobase transporter.