Molecular Biology Unit, National Centre for Cell Science, Pune 411007, India.
BMC Biochem. 2010 Feb 9;11:10. doi: 10.1186/1471-2091-11-10.
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.
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 degrees C and 50 degrees C. Highest ATP hydrolysis activity for all the ATPases was observed in the presence of Mg2+ and Mn2+. However, TraJ and TraK also showed activity in the presence of Co2+. TraJ exhibited the highest specific activity of all the three ATPases with vmax 118 +/- 5.68 nmol/min/mg protein and KM 0.58 +/- 0.10 mM.
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.
四类分泌系统（TFSS）是负责转运各种底物的细菌大分子转运系统，如蛋白质、DNA 或蛋白-DNA 复合物。TFSS 编码两个或三个 ATP 酶，为分泌过程提供能量。这些酶在四类分泌成分中表现出最高的序列保守性。
在这里，我们报告了三种 ATP 酶的生化特性，即 TraE、TraJ 和 TraK（分别为根癌农杆菌转移系统的 VirB4、VirB11 和 VirD4 同源物），来自 Aeromonas veronii 质粒 pAC3249A 的转移系统。ATP 酶在大肠杆菌中以 His 标签融合蛋白的形式表达，并通过亲和层析纯化。用纯化的 ATP 酶进行了 ATP 结合和 ATP 水解实验。TraE 和 TraK 与 TNP-ATP 和 TNP-CTP（分别为 ATP 和 CTP 的荧光类似物）具有很强的结合能力，而 TraJ 则具有较弱的结合能力。三种 ATP 酶的最佳温度范围在 42 度到 50 度之间。所有 ATP 酶在存在 Mg2+和 Mn2+的情况下表现出最高的 ATP 水解活性。然而，TraJ 和 TraK 也在 Co2+存在下表现出活性。TraJ 表现出所有三种 ATP 酶中最高的比活性，vmax 为 118 ± 5.68 nmol/min/mg 蛋白，KM 为 0.58 ± 0.10 mM。
这是首次对 Aeromonas 来源的可接合质粒编码的接合转运 ATP 酶进行生化特征分析。我们的研究表明，A. veronii 质粒 pAc3249A 的新报道的 TFSS 的三种 ATP 酶在 ATP 水解和 ATP 结合方面均具有功能。
