caa a22 4500 445818867 CHVBK 20180317145237.0 cr unu---uuuuu 170323e20110301xx s 000 0 eng 10.1007/s00792-010-0343-2 doi (NATIONALLICENCE)springer-10.1007/s00792-010-0343-2 Identification and characterization of a unique, zinc-containing transport ATPase essential for natural transformation in Thermus thermophilus HB27 [Elektronische Daten] [Ilona Rose, Goran Biuković, Patrick Aderhold, Volker Müller, Gerhard Grüber, Beate Averhoff] Thermus thermophilus is a model strain to unravel the molecular basis of horizontal gene transfer in hot environments. Previous genetic studies led to the identification of a macromolecular transport machinery mediating DNA uptake in an energy-dependent manner. Here, we have addressed how the transporter is energized. Inspection of the genome sequence revealed four putative transport (AAA) ATPases but only the deletion of one, PilF, led to a transformation defect. PilF is similar to transport ATPases of type IV and type II secretions systems but has a unique N-terminal sequence that carries a triplicated GSPII domain. To characterize PilF biochemically it was produced in Escherichia coli and purified. The recombinant protein displayed NTPase activity with a preference for ATP. Gel filtration analyses combined with dynamic light scattering demonstrated that PilF is monodispersed in solution and forms a complex of 590±30kDa, indicating a homooligomer of six subunits. It contains a tetracysteine motif, previously shown to bind Zn2+ in related NTPases. Using atomic absorption spectroscopy, indeed Zn2+ was detected in the enzyme, but in contrast to all known zinc-binding traffic NTPases only one zinc atom was bound to the hexamer. Deletion of the four cysteine residues led to a loss of Zn2+. Nevertheless, the mutant protein retained ATPase activity and hexameric complex formation. Springer, 2011 DNA transporter nationallicence AAA-ATPase nationallicence Metalloproteins nationallicence Molecular motor nationallicence T. thermophilus nationallicence Rose Ilona Molecular Microbiology and Bioenergetics, Institute of Molecular Biosciences, Goethe University, Max-von-Laue-Str. 9, 60438, Frankfurt, Germany aut Biuković Goran Division of Structural and Computational Biology, School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, 637551, Singapore, Republic of Singapore aut Aderhold Patrick Molecular Microbiology and Bioenergetics, Institute of Molecular Biosciences, Goethe University, Max-von-Laue-Str. 9, 60438, Frankfurt, Germany aut Müller Volker Molecular Microbiology and Bioenergetics, Institute of Molecular Biosciences, Goethe University, Max-von-Laue-Str. 9, 60438, Frankfurt, Germany aut Grüber Gerhard Division of Structural and Computational Biology, School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, 637551, Singapore, Republic of Singapore aut Averhoff Beate Molecular Microbiology and Bioenergetics, Institute of Molecular Biosciences, Goethe University, Max-von-Laue-Str. 9, 60438, Frankfurt, Germany aut Extremophiles Springer Japan 15/2(2011-03-01), 191-202 1431-0651 15:2<191 2011 15 792 https://doi.org/10.1007/s00792-010-0343-2 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/s00792-010-0343-2 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Rose Ilona Molecular Microbiology and Bioenergetics, Institute of Molecular Biosciences, Goethe University, Max-von-Laue-Str. 9, 60438, Frankfurt, Germany aut NATIONALLICENCE 700 1- Biuković Goran Division of Structural and Computational Biology, School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, 637551, Singapore, Republic of Singapore aut NATIONALLICENCE 700 1- Aderhold Patrick Molecular Microbiology and Bioenergetics, Institute of Molecular Biosciences, Goethe University, Max-von-Laue-Str. 9, 60438, Frankfurt, Germany aut NATIONALLICENCE 700 1- Müller Volker Molecular Microbiology and Bioenergetics, Institute of Molecular Biosciences, Goethe University, Max-von-Laue-Str. 9, 60438, Frankfurt, Germany aut NATIONALLICENCE 700 1- Grüber Gerhard Division of Structural and Computational Biology, School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, 637551, Singapore, Republic of Singapore aut NATIONALLICENCE 700 1- Averhoff Beate Molecular Microbiology and Bioenergetics, Institute of Molecular Biosciences, Goethe University, Max-von-Laue-Str. 9, 60438, Frankfurt, Germany aut NATIONALLICENCE 773 0- Extremophiles Springer Japan 15/2(2011-03-01), 191-202 1431-0651 15:2<191 2011 15 792 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer