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 1.7 nM, 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.
- Multifunctional enzyme
- Protein membrane associations
- Surface plasmon resonance
ASJC Scopus subject areas
- Molecular Biology