CaiT of Escherichia coli, a new transporter catalyzing L-carnitine/gamma -butyrobetaine exchange.
l-Carnitine is essential for beta-oxidation of fatty acids in mitochondria. Bacterial metabolic pathways are used for the production of this medically important compound. Here, we report the first detailed functional characterization of the caiT gene product, a putative transport protein whose function is required for l-carnitine conversion in Escherichia coli. The caiT gene was overexpressed in E. coli, and the gene product was purified by affinity chromatography and reconstituted into proteoliposomes. Functional analyses with intact cells and proteoliposomes demonstrated that CaiT is able to catalyze the exchange of l-carnitine for gamma-butyrobetaine, the excreted end product of l-carnitine conversion in E. coli, and related betaines. Electrochemical ion gradients did not significantly stimulate l-carnitine uptake. Analysis of l-carnitine counterflow yielded an apparent external K(m) of 105 microm and a turnover number of 5.5 s(-1). Contrary to related proteins, CaiT activity was not modulated by osmotic stress. l-Carnitine binding to CaiT increased the protein fluorescence and caused a red shift in the emission maximum, an observation explained by ligand-induced conformational alterations. The fluorescence effect was specific for betaine structures, for which the distance between trimethylammonium and carboxyl groups proved to be crucial for affinity. Taken together, the results suggest that CaiT functions as an exchanger (antiporter) for l-carnitine and gamma-butyrobetaine according to the substrate/product antiport principle.
Jung
Heinrich
H
Fachbereich Biologie/Chemie, Abteilung Mikrobiologie, Universität Osnabrück, Barbarastrasse 11, D-49069 Osnabrück, Germany. jung_h@biologie.uni-osnabrueck.de
Buchholz
Marion
M
Clausen
Jurgen
J
Nietschke
Monika
M
Revermann
Anne
A
Schmid
Roland
R
Jung
Kirsten
K
D000595:Amino Acid Sequence; D017920:Antiporters / Q000737:chemistry* / Q000378:metabolism*; D001622:Betaine / Q000031:analogs & derivatives* / Q000378:metabolism*; D001692:Biological Transport; D002331:Carnitine / Q000378:metabolism*; D002455:Cell Division; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D008024:Ligands; D008744:Methylamines / Q000378:metabolism; D008954:Models, Biological; D008969:Molecular Sequence Data; D009995:Osmosis; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011510:Proteolipids / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013997:Time Factors
D017920:Antiporters; C469350:CaiT protein, E coli; D029968:Escherichia coli Proteins; D008024:Ligands; D008744:Methylamines; D011510:Proteolipids; C020485:proteoliposomes; D001622:Betaine; C002889:gamma-butyrobetaine; C027451:methylamine; D002331:Carnitine