Miethke Marcus, Klotz Oliver, Linne Uwe, May Jürgen J, Beckering Carsten L, Marahiel Mohamed A
Department of Chemistry, Philipps-Universität Marburg, D-35032 Marburg, Germany.
Mol Microbiol. 2006 Sep;61(6):1413-27. doi: 10.1111/j.1365-2958.2006.05321.x. Epub 2006 Aug 3.
Upon iron limitation, Bacillus subtilis secretes the catecholic trilactone (2,3-dihydroxybenzoate-glycine-threonine)3 siderophore bacillibactin (BB) for ferric iron scavenging. Here, we show that ferri-BB uptake is mediated by the FeuABC transporter and that YuiI, a novel trilactone hydrolase, catalyses ferri-BB hydrolysis leading to cytosolic iron release. Among several Fur-regulated ABC transport mutants, only DeltafeuABC exhibited impaired growth during iron starvation. Quantification of intra- and extracellular (ferri)-BB in iron-depleted DeltafeuABC cultures revealed a fourfold increase of the extracellular siderophore concentration, confirming a blocked ferri-BB uptake in the absence of FeuABC. Ferri-BB was found to bind selectively to the periplasmic binding protein FeuA (Kd = 57 +/- 1 nM), proving high-affinity transport of the iron-charged siderophore. During iron starvation, a DeltayuiI mutant displayed impaired growth and strong intracellular (30-fold) and extracellular (6.5-fold) (ferri)-BB accumulation. Kinetic studies in vitro revealed that YuiI hydrolyses both BB and ferri-BB. While BB hydrolysis led to strong accumulation of the tri- and dimeric reaction intermediates, ferri-BB hydrolysis yielded exclusively the monomeric reaction product and occurred with a 25-fold higher catalytic efficiency than BB single hydrolysis. Thus, ferri-BB was the preferred substrate of the YuiI esterase whose gene locus was designated besA.
在铁限制条件下，枯草芽孢杆菌分泌儿茶酚三内酯（2,3 - 二羟基苯甲酸 - 甘氨酸 - 苏氨酸）3铁载体杆菌铁载体（BB）以清除三价铁。在此，我们表明三价铁 - BB的摄取由FeuABC转运蛋白介导，并且新型三内酯水解酶YuiI催化三价铁 - BB水解，导致胞质铁释放。在几个受Fur调控的ABC转运突变体中，只有ΔfeuABC在铁饥饿期间生长受损。对缺铁的ΔfeuABC培养物中细胞内和细胞外（三价铁） - BB的定量分析显示，细胞外铁载体浓度增加了四倍，证实了在没有FeuABC的情况下三价铁 - BB摄取受阻。发现三价铁 - BB选择性地结合周质结合蛋白FeuA（Kd = 57 +/- 1 nM），证明了带铁的铁载体的高亲和力转运。在铁饥饿期间，ΔyuiI突变体生长受损，细胞内（30倍）和细胞外（6.5倍）（三价铁） - BB强烈积累。体外动力学研究表明，YuiI水解BB和三价铁 - BB。虽然BB水解导致三聚体和二聚体反应中间体的强烈积累，但三价铁 - BB水解仅产生单体反应产物，并且催化效率比BB单次水解高25倍。因此，三价铁 - BB是YuiI酯酶的优选底物，其基因座被指定为besA。
