Conformational change and membrane association of the PutA protein are coincident with reduction of its FAD cofactor by proline.
The PutA protein is both the put repressor and a membrane-bound enzyme with proline and delta 1-pyrroline-5-carboxylate dehydrogenase activities. The conditions required for association of purified PutA protein with membrane vesicles suggested that a redox switching mechanism might determine the proportion of PutA protein functioning as a dehydrogenase (Wood, J. M. (1987) Proc. Natl. Acad. Sci. USA 84, 373-377). The FAD cofactor was released from the PutA protein with 1 M KBr at neutral pH. The apoprotein retained delta 1-pyrroline-5-carboxylate dehydrogenase and DNA binding but not proline dehydrogenase activity. Reconstitution with FAD fully restored proline dehydrogenase activity. Proline at a concentration of 0.11 mM caused half-maximal bleaching of the FAD in PutA. Chymotryptic digestion of the PutA protein in the presence and absence of proline demonstrated that the persistence of a 119-kDa protein fragment was characteristic of the reduced protein. Identical digestion patterns were obtained from the apoprotein in the presence and absence of proline. The quantity of the 119-kDa fragment produced varied with proline concentration, yielding a midpoint of 0.056 mM proline. The fraction of PutA protein associated with membrane vesicles was also a function of proline concentration, yielding a titration midpoint of 0.10 mM proline. Membrane binding was thus coincident with both flavin reduction and a change in protein conformation.
Brown
E D
ED
Guelph-Waterloo Centre for Graduate Work in Chemistry, Department of Microbiology, University of Guelph, Ontario, Canada.
Wood
J M
JM
D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D002918:Chymotrypsin; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D010084:Oxidation-Reduction; D011392:Proline / Q000378:metabolism*; D011487:Protein Conformation
D001426:Bacterial Proteins; D008565:Membrane Proteins; C080683:PutA protein, Bacteria; D005182:Flavin-Adenine Dinucleotide; D011392:Proline; D002918:Chymotrypsin