Jung Heinrich, Buchholz Marion, Clausen Jurgen, Nietschke Monika, Revermann Anne, Schmid Roland, Jung Kirsten
Fachbereich Biologie/Chemie, Abteilung Mikrobiologie, Universität Osnabrück, Barbarastrasse 11, D-49069 Osnabrück, Germany.
J Biol Chem. 2002 Oct 18;277(42):39251-8. doi: 10.1074/jbc.M206319200. Epub 2002 Aug 5.
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.
左旋肉碱对于线粒体中脂肪酸的β氧化至关重要。细菌代谢途径被用于生产这种具有医学重要性的化合物。在此，我们报告了caiT基因产物的首次详细功能表征，该产物是一种假定的转运蛋白，其功能是大肠杆菌中左旋肉碱转化所必需的。caiT基因在大肠杆菌中过表达，基因产物通过亲和层析纯化并重组到蛋白脂质体中。对完整细胞和蛋白脂质体的功能分析表明，CaiT能够催化左旋肉碱与γ-丁甜菜碱（大肠杆菌中左旋肉碱转化的排泄终产物）以及相关甜菜碱的交换。电化学离子梯度并未显著刺激左旋肉碱的摄取。左旋肉碱逆流分析得出表观外部米氏常数为105微摩尔，周转数为5.5秒⁻¹。与相关蛋白不同，CaiT活性不受渗透胁迫调节。左旋肉碱与CaiT的结合增加了蛋白质荧光并导致发射最大值出现红移，这一观察结果可由配体诱导的构象改变来解释。荧光效应对于甜菜碱结构具有特异性，事实证明三甲基铵和羧基之间的距离对于亲和力至关重要。综上所述，结果表明CaiT根据底物/产物反向转运原理作为左旋肉碱和γ-丁甜菜碱的交换体（反向转运体）发挥作用。
