Kinetic and thermodynamic analysis of Bradyrhizobium japonicum PutA-membrane associations.
Kinetic and thermodynamic analysis of Bradyrhizobium japonicum PutA-membrane associations.
复制标题
日本慢生根瘤菌 PutA 膜关联的动力学和热力学分析。
DOI：
10.1016/j.abb.2005.10.022
复制
发表时间：
2006
期刊：
影响因子：
--
通讯作者：
Becker,DonaldF
中科院分区：
文献类型：
--
作者：
Zhang,Weimin;Krishnan,Navasona;Becker,DonaldF
在大肠杆菌中，脯氨酸诱导PutA黄素酶的紧密膜结合，并将PutA从转录阻遏物转化为膜相关脯氨酸分解代谢酶。在其他革兰氏阴性细菌如慢生根瘤菌中，PutA缺乏DNA结合活性，仅作为脯氨酸分解代谢酶发挥作用。在这里，我们从B中表征PutA的膜结合性质。日本根瘤菌（BjPutA）的研究，以解决脯氨酸是否类似于大肠杆菌PutA（EcPutA）调节BjPutA-脂质结合。BjPutA-脂质结合的表面等离子体共振（SPR）动力学测量显示，BjPutA在不存在和存在脯氨酸的情况下与脂质形成复合物，其解离常数（KD）值分别为2.5和1.7 nM。使用不同电荷的脂质双层的SPR实验表明BjPutA选择性地结合带负电荷的脂质，这与EcPutA的电荷独立的膜结合形成对比。在25°C下通过等温滴定量热法分析BjPutA-脂质结合揭示了熵驱动的吸热结合反应。这项工作表明，BjPutA膜协会显着不同的EcPutA。
In Escherichia coli, proline induces tight membrane binding of the PutA flavoenzyme and transforms PutA from a transcriptional repressor to a membrane-associated proline catabolic enzyme. In other gram-negative bacteria such as Bradyrhizobium japonicum, PutA lacks DNA binding activity and functions only as a proline catabolic enzyme. Here, we characterize the membrane binding properties of PutA from B. japonicum (BjPutA) to address whether proline regulates BjPutA–lipid binding similar to Escherichia coli PutA (EcPutA). Surface plasmon resonance (SPR) kinetic measurements of BjPutA–lipid binding show BjPutA forms a complex with lipids in the absence and presence of proline with similar dissociation constant (KD) values of 2.5 and 1.7nM, respectively. SPR experiments using differently charged lipid bilayers indicate BjPutA selectively binds negatively charged lipids, which contrasts with the charge independent membrane binding of EcPutA. Analysis of BjPutA–lipid binding by isothermal titration calorimetry at 25°C revealed an endothermic binding reaction that is entropically driven. This work shows that BjPutA–membrane associations vary significantly from EcPutA.