Recognition of ferric catecholates by FepA
Recognition of ferric catecholates by FepA
复制标题
DOI：
10.1128/jb.186.11.3578-3589.2004
复制
发表时间：
2004-06-01
影响因子：
3.2
通讯作者：
Klebba, PE
中科院分区：
文献类型：
--
作者：
Annamalai, R;Jin, B;Klebba, PE
大肠杆菌FepA转运某些儿茶酚铁载体，但不转运其他铁载体，也不转运任何非儿茶酚化合物。直接结合和竞争实验表明，这种选择性起源于吸附阶段。合成的三儿茶酚盐Fe-TRENCAM与FepA的结合亲和力比Fe-肠杆菌素（FeEnt）低50至100倍，尽管具有相同的金属中心，并且Fe-棒状杆菌素仅在高得多的浓度下结合。无论是铁-agrobactin或ferrichrotne结合在所有，即使在浓度10（6）倍以上的K-d因此，FepA只吸附儿茶酚铁络合物，它选择FeEnt之间，甚至其密切的同系物。我们使用丙氨酸扫描诱变来研究表面芳香残基对FeEnt识别的贡献。虽然从晶体学上看不明显，但L3、L5、L7、L 8和L10中的芳香族残基影响FepA与FeEnt的相互作用。在消除芳香族残基的10种取代中，K-d增加多达20倍（Y 481 A和Y 638 A），K-m增加多达400倍（Y 478），表明孔入口周围芳香性的重要性。虽然许多突变同样减少了结合和运输，但其他突变导致后者更大的缺陷。Y 638 A和Y 478 A使K-m分别比K-d增加10倍和200倍。N-结构域环缺失产生相同的表型：Delta 60 -67（在NL 1中）和Delta 98 -105（在NL 2中）使K-d增加10至20倍，但使K-m增加500至700倍。W101 A（在NL 2中）对K-d几乎没有影响，但使K-m增加了1,000倍。这些数据表明，N末端的主要作用是在配体摄取。荧光和放射性同位素实验显示FeEnt从FepA的双相释放。在光谱测定中，k（off 1）为0.03/s，k（off 2）为0.003/s。然而，FepAY 272 AF 329 A没有表现出快速解离相，证实了芳香族残基在FeEnt的初始结合中的作用。因此，β-桶环包含主要的配体识别决定簇，N-结构域环在配体转运中发挥作用。
Escherichia coli FepA transports certain catecholate ferric siderophores, but not others, nor any noncatecholate compounds. Direct binding and competition experiments demonstrated that this selectivity originates during the adsorption stage. The synthetic tricatecholate Fe-TRENCAM bound to FepA with 50- to 100-fold-lower affinity than Fe-enterobactin (FeEnt), despite an identical metal center, and Fe-corynebactin only bound at much higher concentrations. Neither Fe-agrobactin nor ferrichrotne bound at all, even at concentrations 10(6)-fold above the K-d Thus, FepA only adsorbs catecholate iron complexes, and it selects FeEnt among even its close homologs. We used alanine scanning mutagenesis to study the contributions of surface aromatic residues to FeEnt recognition. Although not apparent from crystallography, aromatic residues in L3, L5, L7, L8, and L10 affected FepA's interaction with FeEnt. Among 10 substitutions that eliminated aromatic residues, K-d increased as much as 20-fold (Y481A and Y638A) and K-m increased as much as 400-fold (Y478), showing the importance of aromaticity around the pore entrance. Although many mutations equally reduced binding and transport, others caused greater deficiencies in the latter. Y638A and Y478A increased K-m 10- and 200-fold more, respectively, than K-d. N-domain loop deletions created the same phenotype: Delta60-67 (in NL1) and Delta98-105 (in NL2) increased K-d 10- to 20-fold but raised K-m 500- to 700-fold. W101A (in NL2) had little effect on K-d but increased K-m 1,000-fold. These data suggested that the primary role of the N terminus is in ligand uptake. Fluorescence and radioisotopic experiments showed biphasic release of FeEnt from FepA. In spectroscopic determinations, k(off1) was 0.03/s and k(off2) was 0.003/s. However, FepAY272AF329A did not manifest the rapid dissociation phase, corroborating the role of aromatic residues in the initial binding of FeEnt. Thus, the beta-barrel loops contain the principal ligand recognition determinants, and the N-domain loops perform a role in ligand transport.