Structure and mechanism of Pseudomonas aeruginosa PhzD, an isochorismatase from the phenazine biosynthetic pathway
Structure and mechanism of Pseudomonas aeruginosa PhzD, an isochorismatase from the phenazine biosynthetic pathway
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DOI：
10.1021/bi027385d
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发表时间：
2003-05-20
期刊：
影响因子：
2.9
通讯作者：
Ladner, JE
中科院分区：
文献类型：
--
作者：
Parsons, JF;Calabrese, K;Ladner, JE
来自铜绿假单胞菌的PhzD是一种参与吩嗪生物合成的异分支酶。吩嗪类是一种抗菌化合物，在某些环境中为假单胞菌提供竞争优势，可能是假单胞菌感染持续存在的部分原因。在体内，PhzD催化2-氨基-2-脱氧异分支酸酯的乙烯基醚官能团的水解，产生丙酮酸和反式-2，3-二氢-3-羟基邻氨基苯甲酸，其随后用于吩嗪生物合成途径。PhzD还催化相关的乙烯基醚异分支酸酯、分支酸酯和4-氨基-4-脱氧分支酸酯的水解。在这里，我们报告了天然PhzD的1.5埃晶体结构，以及与异分支酸盐复合的无活性D38 A变体的1.6埃结构。结构揭示了异分支酸酯以反式双轴构象结合到PhzD活性位点，并且结构的叠加表明异分支酸酯的亚甲基N-乙酰基碳与天冬氨酸酯38的侧链羧酸酯相邻。天冬氨酸38与异分支酸的接近和由天冬氨酸38转化为丙氨酸导致的活性的完全丧失表明了一种机制，其中羧酸盐充当一般酸以使底物质子化，产生碳阳离子/氧碳鎓离子，然后其快速水合以形成半缩酮中间体，然后其自发分解为产物。PhzD的结构与α/β-水解酶亚家族的其他结构非常相似，包括吡嗪酰胺酶和N-氨基甲酰基肌氨酸酰胺水解酶。然而，PhzD催化不相关的化学反应，并且缺乏在其结构上密切相关的亲核半胱氨酸。由PhzD催化的乙烯基醚水解代表在α/β-水解酶家族中看到的催化多样性的又一个实例，其成员也已知水解酰胺、磷酸酯、膦酸酯、环氧化物和C-X键。
PhzD from Pseudomonas aeruginosa is an isochorismatase involved in phenazine biosynthesis. Phenazines are antimicrobial compounds that provide Pseudomonas with a competitive advantage in certain environments and may be partly responsible for the persistence of Pseudomonas infections. In vivo, PhzD catalyzes the hydrolysis of the vinyl ether functional group of 2-amino-2-deoxyisochorismate, yielding pyruvate and trans-2,3-dihydro-3-hydroxyanthranilic acid, which is then utilized in the phenazine biosynthetic pathway. PhzD also catalyzes hydrolysis of the related vinyl ethers isochorismate, chorismate, and 4-amino-4-deoxychorismate. Here we report the 1.5 Angstrom crystal structure of native PhzD, and the 1.6 Angstrom structure of the inactive D38A variant in complex with isochorismate. The structures reveal that isochorismate binds to the PhzD active site in a trans-diaxial conformation, and superposition of the structures indicates that the methylene pyruvyl carbon of isochorismate is adjacent to the side chain carboxylate of aspartate 38. The proximity of aspartate 38 to isochorismate and the complete loss of activity resulting from the conversion of aspartate 38 to alanine suggest a mechanism in which the carboxylate acts as a general acid to protonate the substrate, yielding a carbocation/oxocarbonium ion that is then rapidly hydrated to form a hemiketal intermediate, which then decomposes spontaneously to products. The structure of PhzD is remarkably similar to other structures from a subfamily of alpha/beta-hydrolase enzymes that includes pyrazinamidase and N-carbamoylsarcosine amidohydrolase. However, PhzD catalyzes unrelated chemistry and lacks a nucleophilic cysteine found in its close structural relatives. The vinyl ether hydrolysis catalyzed by PhzD represents yet another example of the catalytic diversity seen in the alpha/beta-hydrolase family, whose members are also known to hydrolyze amides, phosphates, phosphonates, epoxides, and C-X bonds.