Relationships between Heme Incorporation, Tetramer Formation, and Catalysis of a Heme-regulated Phosphodiesterase from Escherichia coli
Relationships between Heme Incorporation, Tetramer Formation, and Catalysis of a Heme-regulated Phosphodiesterase from Escherichia coli
复制标题
DOI：
10.1074/jbc.m304408200
复制
发表时间：
2003-12
影响因子：
4.8
通讯作者：
Tokiko Yoshimura;I. Sagami;Yukie Sasakura;Toru Shimizu
中科院分区：
文献类型：
--
作者：
Tokiko Yoshimura;I. Sagami;Yukie Sasakura;Toru Shimizu
来自大肠杆菌（Ec DOS）的血红素调节磷酸二酯酶（PDE）是一种四聚体蛋白，由一个n端传感器结构域（氨基酸1-201）和一个c端催化结构域（氨基酸336-799）组成，该结构域包含两个PAS结构域（氨基酸21-84和144-201）。血红素与PAS-A结构域结合，血红素铁的氧化还原状态调节PDE活性。在我们的实验中，H77A突变和PAS-B结构域的缺失导致血红素与PAS-A结合亲和力的丧失。然而，两种突变蛋白仍然是四聚体的，并且比全长野生型酶更有活性（比全长野生型酶活性高140%），这表明血红素结合对催化不是必需的。一个不含PAS-A结构域或血红素的n端截断突变体（ΔN147，氨基酸148-807）与全长野生型蛋白相比，显示出160%的活性，证实血红素结合PAS-A结构域不是催化活性所必需的。对c端截断突变体的分析导致了负责四聚体形成的区域的定位，并揭示了PDE仅在四聚体蛋白中具有活性。假设的金属离子结合位点（His-590, His-594）的突变完全破坏了PDE活性，表明Mg2+与该位点的结合对催化作用至关重要。有趣的是，在全长野生型蛋白中加入Fe2+形式的分离PAS-A结构域显著提高了PDE活性（>5倍）。这种活化可能是由于分离的PAS-A结构域与全酶结构域之间的相互作用导致催化位点的结构变化。
The heme-regulated phosphodiesterase (PDE) from Escherichia coli (Ec DOS) is a tetrameric protein composed of an N-terminal sensor domain (amino acids 1-201) containing two PAS domains (PAS-A, amino acids 21-84, and PAS-B, amino acids 144-201) and a C-terminal catalytic domain (amino acids 336-799). Heme is bound to the PAS-A domain, and the redox state of the heme iron regulates PDE activity. In our experiments, a H77A mutation and deletion of the PAS-B domain resulted in the loss of heme binding affinity to PAS-A. However, both mutant proteins were still tetrameric and more active than the full-length wild-type enzyme (140% activity compared with full-length wild type), suggesting that heme binding is not essential for catalysis. An N-terminal truncated mutant (ΔN147, amino acids 148-807) containing no PAS-A domain or heme displayed 160% activity compared with full-length wild-type protein, confirming that the heme-bound PAS-A domain is not required for catalytic activity. An analysis of C-terminal truncated mutants led to mapping of the regions responsible for tetramer formation and revealed PDE activity in tetrameric proteins only. Mutations at a putative metal-ion binding site (His-590, His-594) totally abolished PDE activity, suggesting that binding of Mg2+ to the site is essential for catalysis. Interestingly, the addition of the isolated PAS-A domain in the Fe2+ form to the full-length wild-type protein markedly enhanced PDE activity (>5-fold). This activation is probably because of structural changes in the catalytic site as a result of interactions between the isolated PAS-A domain and that of the holoenzyme.