TY - JOUR
T1 - Kinetic and thermodynamic analysis of Bradyrhizobium japonicum PutA-membrane associations
AU - Zhang, Weimin
AU - Krishnan, Navasona
AU - Becker, Donald F.
N1 - Funding Information:
This research was supported in part by NSF MCB0340912, NIH GM061068, University of Nebraska Biochemistry Department and Redox Biology Center, and the Nebraska Agricultural Research Division, Journal Series No. 14581. This publication was also made possible by NIH Grant No. P20 RR-017675-02 from the National Center for Research Resources. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIH.
PY - 2006/1/1
Y1 - 2006/1/1
N2 - 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.
AB - 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.
KW - Flavoprotein
KW - Multifunctional enzyme
KW - Protein membrane associations
KW - Surface plasmon resonance
UR - http://www.scopus.com/inward/record.url?scp=29244452111&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=29244452111&partnerID=8YFLogxK
U2 - 10.1016/j.abb.2005.10.022
DO - 10.1016/j.abb.2005.10.022
M3 - Article
C2 - 16310755
AN - SCOPUS:29244452111
SN - 0003-9861
VL - 445
SP - 174
EP - 183
JO - Archives of Biochemistry and Biophysics
JF - Archives of Biochemistry and Biophysics
IS - 1
ER -