A homodimer represents an active species of the peptidyl-prolyl cis/trans isomerase FKBP25mem from Legionella pneumophila.
The molecular mass of the native FK506-binding peptidyl-prolyl cis/trans isomerase (PPIase) FKBP25mem from Legionella pneumophila (Mip (macrophage infectivity potentiator) protein) was determined by two methods. By gel-permeation chromatography we found no indication of the presence of the monomeric enzyme. However, an oligomeric state with a molecular mass of about 62 kDa was detected. By cross-linking with dimethyl pimelimidate and subsequent SDS-PAGE of either the surface proteins of intact L. pneumophila cells or the purified recombinant FKBP25mem in solution, we observed an immunoreactive band indicative of a mass in the dimer range. In contrast to human recombinant FKBP12, the enzymatic activity of Legionella FKBP25men was strongly dependent on the protein concentration, pointing to a dimer as the most active species. However, the inhibition by FK506 yielded a nearly constant value of Ki of about 250 nM when measured in the same range of FKBP25mem concentration. These results may be explained by the fact that monomeric FKBP25mem has little, if any, influence on enzymatic activity when compared with the homodimer.
Schmidt
B
B
Max-Planck-Gesellschaft, Arbeitsgruppe Enzymologie der Peptidbindung, Halle/Saale, Germany.
Rahfeld
J
J
Schierhorn
A
A
Ludwig
B
B
Hacker
J
J
Fischer
G
G
D000591:Amino Acid Isomerases / Q000737:chemistry* / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D002473:Cell Wall / Q000378:metabolism; D002850:Chromatography, Gel; D003432:Cross-Linking Reagents; D006360:Heat-Shock Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry; D006801:Humans; D007096:Imidoesters; D020104:Immunophilins; D007700:Kinetics; D016952:Legionella pneumophila / Q000201:enzymology*; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D008970:Molecular Weight; D019696:Peptidylprolyl Isomerase; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016559:Tacrolimus / Q000494:pharmacology; D022021:Tacrolimus Binding Proteins
D001426:Bacterial Proteins; D002352:Carrier Proteins; D003432:Cross-Linking Reagents; D006360:Heat-Shock Proteins; D007096:Imidoesters; D008565:Membrane Proteins; D011994:Recombinant Proteins; C029326:dimethyl pimelimidate; D000591:Amino Acid Isomerases; D022021:Tacrolimus Binding Proteins; D020104:Immunophilins; C512922:Mip protein, Legionella pneumophila; D019696:Peptidylprolyl Isomerase; D016559:Tacrolimus