RESUMO
Bacteria, as well as the plastid organelles of algae and higher plants, utilize proteins of the suf operon. These are involved in Fe-S cluster assembly, particularly under conditions of iron limitation or oxidative stress. Genetic experiments in some organisms found that the ATPase SufC is essential, though its role in Fe-S biogenesis remains unclear. To ascertain how interactions with other individual Suf proteins affect the activity of SufC we coexpressed it with either SufB or SufD from Thermotoga maritima and purified the resulting SufBC and SufCD complexes. Analytical ultracentrifuge and multiangle light-scattering measurements showed that the SufBC complex exists in solution as the tetrameric SufB(2)C(2) species, whereas SufCD exists as an equilibrium mixture of SufCD and SufC(2)D(2). Transient kinetic studies of the complexes were made using fluorescent 2'(3')-O-(N-methylanthraniloyl-(mant) analogues of ATP and ADP. Both SufBC and SufCD bound mantATP and mantADP much more tightly than does SufC alone. Compared to the cleavage step of the mantATPase of SufC alone, that of SufBC was accelerated 180-fold and that of SufCD only fivefold. Given that SufB and SufD have 20% sequence identity and similar predicted secondary structures, the different hydrodynamic properties and kinetic mechanisms of the two complexes are discussed.
Assuntos
Difosfato de Adenosina/química , Trifosfato de Adenosina/química , Proteínas de Bactérias/química , Eletroforese em Gel de Poliacrilamida , Corantes Fluorescentes , Cinética , Espectrometria de FluorescênciaRESUMO
Protein products of the suf operon are involved in iron-sulfur metabolism. SufC is an ATPase that can interact with SufB in the absence of nucleotide. We have studied the transient kinetics of the SufC ATPase mechanism using the fluorescent ATP analogue, 2'(3')-O-N-methylanthraniloyl-ATP (mantATP). mantATP initially binds to SufC weakly. A conformational change of the SufC.mantATP complex then occurs followed by the very slow cleavage of mantATP to mantADP and the rapid release of Pi. In the presence of SufB, the cleavage step is accelerated and the release of mantADP is inhibited. Both of these effects promote the formation of a SufC.mantADP complex. In the absence and presence of SufB, mantADP remains more tightly bound to SufC than mantATP. These studies provide a basis for how the SufB and -C proteins interact in the processes involved in regulating iron-sulfur transfer.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/genética , Difosfato de Adenosina/análogos & derivados , Difosfato de Adenosina/genética , Difosfato de Adenosina/metabolismo , Adenosina Trifosfatases/genética , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/genética , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/metabolismo , Cromatografia Líquida de Alta Pressão , Escherichia coli/genética , Polarização de Fluorescência , Corantes Fluorescentes/metabolismo , Proteínas Ferro-Enxofre/genética , Proteínas Ferro-Enxofre/metabolismo , Cinética , Óperon , Fósforo/metabolismo , Conformação Proteica , Thermotoga maritima/química , ortoaminobenzoatos/metabolismoRESUMO
The replication and repair of organellar genomes in the malaria parasite Plasmodium falciparum is poorly understood. We have assessed the properties of an open reading frame Pfprex (formerly known as pom1) and confirm that it specifies a multi-domain polypeptide with DNA primase, DNA helicase, DNA polymerase and 3'-5' exonuclease activities. The sequence of the primase/helicase domain is phylogenetically related to the T7-bacteriophage gene 4 product and mammalian mitochondrial helicase, Twinkle. Despite that, the N-terminal sequence of this multi-domain polypeptide directs a green fluorescent protein reporter specifically to the P. falciparum apicoplast and not to the mitochondrion. Phylogenetic analysis placed the DNA polymerase sequence with the family A bacterial polymerases, most closely to those of the thermophilic Aquifex species. Notably, the malarial enzyme was optimally active at 75 degrees C. Pfprex is the first example of a gene encoding contiguous DNA polymerase, DNA primase and DNA helicase components. We propose it has a key role in replication of the malarial plastid genome, a validated drug target.
Assuntos
Replicação do DNA , Complexos Multienzimáticos/genética , Organelas/metabolismo , Plasmodium falciparum/enzimologia , Proteínas de Protozoários/genética , Animais , DNA Helicases/química , DNA Helicases/genética , DNA Helicases/metabolismo , DNA Polimerase III/química , DNA Polimerase III/genética , DNA Polimerase III/metabolismo , DNA Primase/química , DNA Primase/genética , DNA Primase/metabolismo , Exonucleases/química , Exonucleases/genética , Exonucleases/metabolismo , Genes de Protozoários , Genes Reporter , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Complexos Multienzimáticos/química , Complexos Multienzimáticos/metabolismo , Organelas/genética , Filogenia , Plasmodium falciparum/genética , Estrutura Terciária de Proteína , Transporte Proteico , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Homologia de SequênciaRESUMO
Genetic experiments in bacteria have shown the suf operon is involved in iron homeostasis and the oxidative stress response. The sufB and sufC genes that always occur together in bacteria are also found in plants, and even the malaria parasite, associated with the plastid organelle. Although the suf operon is believed to encode an iron-dependent ABC-transporter there is no direct evidence. By immunolocalization we show here that SufB and SufC are associated with the membrane of Escherichia coli. We also present kinetic studies with a recombinant version of SufC from Thermotoga maritima that shows it is an ATPase and that it interacts with SufB in vitro.