Biochemical characterization of three putative ATPases from a new type IV secretion system of Aeromonas veronii plasmid pAC3249A
Background Type four secretion systems (TFSS) are bacterial macromolecular transport systems responsible for transfer of various substrates such as proteins, DNA or protein-DNA complexes. TFSSs encode two or three ATPases generating energy for the secretion process. These enzymes exhibit highest sequence conservation among type four secretion components. Results Here, we report the biochemical characterization of three ATPases namely TraE, TraJ and TraK (VirB4, VirB11 and VirD4 homologs of the Agrobacterium tumefaciens transfer system, respectively) from the transfer system of Aeromonas veronii plasmid pAC3249A. ATPases were expressed as His-tag fusion proteins in E. coli and purified by affinity chromatography. ATP binding and ATP hydrolysis experiments were performed with the purified ATPases. TraE and TraK showed strong binding to TNP-ATP and TNP-CTP (fluorescent analogs of ATP and CTP respectively) whereas TraJ showed weak binding. The optimum temperature range for the three ATPases was between 42°C and 50°C. Highest ATP hydrolysis activity for all the ATPases was observed in the presence of $ Mg^{2+} $ and $ Mn^{2+} $. However, TraJ and TraK also showed activity in the presence of $ Co^{2+} $. TraJ exhibited the highest specific activity of all the three ATPases with $ v_{max} $ 118 ± 5.68 nmol/min/mg protein and $ K_{M} $ 0.58 ± 0.10 mM. Conclusions This is the first biochemical characterization of conjugative transport ATPases encoded by a conjugative plasmid from Aeromonas. Our study demonstrated that the three ATPases of a newly reported TFSS of A. veronii plasmid pAc3249A are functional in both ATP hydrolysis and ATP binding..
| Medienart: |
E-Artikel |
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| Erscheinungsjahr: |
2010 |
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| Erschienen: |
2010 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:11 |
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| Enthalten in: |
BMC biochemistry - 11(2010), 1 vom: 09. Feb. |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
Rangrez, Ashraf Y [VerfasserIn] |
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| Links: |
Volltext [lizenzpflichtig] |
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| Themen: |
ATPase Activity |
| Anmerkungen: |
© Rangrez et al; licensee BioMed Central Ltd. 2010. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( |
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| doi: |
10.1186/1471-2091-11-10 |
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| PPN (Katalog-ID): |
OLC2100830740 |
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| 100 | 1 | |a Rangrez, Ashraf Y |e verfasserin |4 aut | |
| 245 | 1 | 0 | |a Biochemical characterization of three putative ATPases from a new type IV secretion system of Aeromonas veronii plasmid pAC3249A |
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| 500 | |a © Rangrez et al; licensee BioMed Central Ltd. 2010. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( | ||
| 520 | |a Background Type four secretion systems (TFSS) are bacterial macromolecular transport systems responsible for transfer of various substrates such as proteins, DNA or protein-DNA complexes. TFSSs encode two or three ATPases generating energy for the secretion process. These enzymes exhibit highest sequence conservation among type four secretion components. Results Here, we report the biochemical characterization of three ATPases namely TraE, TraJ and TraK (VirB4, VirB11 and VirD4 homologs of the Agrobacterium tumefaciens transfer system, respectively) from the transfer system of Aeromonas veronii plasmid pAC3249A. ATPases were expressed as His-tag fusion proteins in E. coli and purified by affinity chromatography. ATP binding and ATP hydrolysis experiments were performed with the purified ATPases. TraE and TraK showed strong binding to TNP-ATP and TNP-CTP (fluorescent analogs of ATP and CTP respectively) whereas TraJ showed weak binding. The optimum temperature range for the three ATPases was between 42°C and 50°C. Highest ATP hydrolysis activity for all the ATPases was observed in the presence of $ Mg^{2+} $ and $ Mn^{2+} $. However, TraJ and TraK also showed activity in the presence of $ Co^{2+} $. TraJ exhibited the highest specific activity of all the three ATPases with $ v_{max} $ 118 ± 5.68 nmol/min/mg protein and $ K_{M} $ 0.58 ± 0.10 mM. Conclusions This is the first biochemical characterization of conjugative transport ATPases encoded by a conjugative plasmid from Aeromonas. Our study demonstrated that the three ATPases of a newly reported TFSS of A. veronii plasmid pAc3249A are functional in both ATP hydrolysis and ATP binding. | ||
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