Crystal structure of the pyocyanin biosynthetic protein PhzS
Crystal structure of the pyocyanin biosynthetic protein PhzS
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DOI：
10.1021/bi702480t
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发表时间：
2008-05-13
期刊：
影响因子：
2.9
通讯作者：
Parsons, James F.
中科院分区：
文献类型：
--
作者：
Greenhagen, Bryan T.;Shi, Katherine;Parsons, James F.
人类病原体铜绿假单胞菌产生绿脓菌素，一种蓝色的吩嗪衍生物，已知其在毒力中起作用。绿脓菌素是由氯酸通过吩嗪途径产生的，由一个基因簇编码的九种蛋白质。吩嗪-1-羧酸，最初形成的吩嗪，在两个步骤中转化为绿脓菌素，这两个步骤由酶PhzM和PhzS催化。PhzM是腺苷甲硫氨酸依赖性甲基转移酶，PhzS是黄素依赖性羟化酶。已经表明，PhzM仅在PhzS的物理存在下才有活性，这表明蛋白质-蛋白质相互作用参与绿脓菌素的形成。这样的复合物将阻止5-甲基-吩嗪-1-羧酸酯的释放，这是一种推定的中间体，并且是一种明显不稳定的化合物。在这里，我们描述的三维结构的PhzS，解决了单一的异常色散，在2.4埃的分辨率。结构表明PhzS是以对羟基苯甲酸羟化酶为特征的芳香族羟化酶家族的成员。PhzS的黄素辅因子在溶剂中暴露于通常在未配体的芳香族羟化酶中看到的取向。PhzS黄素，然而，似乎是由堆叠相互作用和氢键的组合在一个紧张的构象举行。该结构表明，进入活性位点是通过蛋白质相对侧的隧道获得的，从黄素暴露的地方。C-末端23个残基是无序的，因为这些原子不存在电子密度。C-末端的可能位置，靠近底物通道，表明它可能参与底物结合，如另一种结构同源物RebC所示。该区域也可以是PhzM-PhzS界面的元件。芳香族羟化酶已被证明可以催化活化底物上的亲电取代反应。然而，假定的PhzS底物是缺电子的，不太可能充当亲核试剂，这表明PhzS可能使用与其结构亲属不同的机制。
The human pathogen Pseudomonas aeruginosa produces pyocyanin, a blue-pigmented phenazine derivative, which is known to play a role in virulence. Pyocyanin is produced from chorismic acid via the phenazine pathway, nine proteins encoded by a gene cluster. Phenazine-l-carboxylic acid, the initial phenazine formed, is converted to pyocyanin in two steps that are catalyzed by the enzymes PhzM and PhzS. PhzM is an adenosylmethionine dependent methyltransferase, and PhzS is a flavin dependent hydroxylase. It has been shown that PhzM is only active in the physical presence of PhzS, suggesting that a protein-protein interaction is involved in pyocyanin formation. Such a complex would prevent the release of 5-methyl-phenazine-1-carboxylate, the putative intermediate, and an apparently unstable compound. Here, we describe the three-dimensional structure of PhzS, solved by single anomalous dispersion, at a resolution of 2.4 angstrom. The structure reveals that PhzS is a member of the family of aromatic hydroxylases characterized by p-hydroxybenzoate hydroxylase. The flavin cofactor of PhzS is in the solvent exposed out orientation typically seen in unliganded aromatic hydroxylases. The PhzS flavin, however, appears to be held in a strained conformation by a combination of stacking interactions and hydrogen bonds. The structure suggests that access to the active site is gained via a tunnel on the opposite side of the protein from where the flavin is exposed. The C-terminal 23 residues are disordered as no electron density is present for these atoms. The probable location of the C-terminus, near the substrate access tunnel, suggests that it may be involved in substrate binding as has been shown for another structural homologue, RebC. This region also may be an element of a PhzM-PhzS interface. Aromatic hydroxylases have been shown to catalyze electrophilic substitution reactions on activated substrates. The putative PhzS substrate, however, is electron deficient and unlikely to act as a nucleophile, suggesting that PhzS may use a different mechanism than its structural relatives.