Structure and Mechanism of Pseudomonas aeruginosa PhzD, an Isochorismatase from the Phenazine Biosynthetic Pathway
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- 作者：James F. Parsons ; Kelly Calabrese ; Edward Eisenstein ; and Jane E. Ladner
- 刊名：Biochemistry
- 出版年：2003
- 出版时间：May 20, 2003
- 年：2003
- 卷：42
- 期：19
- 页码：5684 - 5693
- 全文大小：427K
- 年卷期：v.42,no.19(May 20, 2003)
- ISSN：1520-4995
文摘
PhzD from Pseudomonas aeruginosa is an isochorismatase involved in phenazine biosynthesis.Phenazines are antimicrobial compounds that provide Pseudomonas with a competitive advantage in certainenvironments and may be partly responsible for the persistence of Pseudomonas infections. In vivo, PhzDcatalyzes the hydrolysis of the vinyl ether functional group of 2-amino-2-deoxyisochorismate, yieldingpyruvate and trans-2,3-dihydro-3-hydroxyanthranilic acid, which is then utilized in the phenazinebiosynthetic pathway. PhzD also catalyzes hydrolysis of the related vinyl ethers isochorismate, chorismate,and 4-amino-4-deoxychorismate. Here we report the 1.5 Å crystal structure of native PhzD, and the 1.6Å structure of the inactive D38A variant in complex with isochorismate. The structures reveal thatisochorismate binds to the PhzD active site in a trans-diaxial conformation, and superposition of thestructures indicates that the methylene pyruvyl carbon of isochorismate is adjacent to the side chaincarboxylate of aspartate 38. The proximity of aspartate 38 to isochorismate and the complete loss ofactivity resulting from the conversion of aspartate 38 to alanine suggest a mechanism in which thecarboxylate acts as a general acid to protonate the substrate, yielding a carbocation/oxocarbonium ionthat is then rapidly hydrated to form a hemiketal intermediate, which then decomposes spontaneously toproducts. The structure of PhzD is remarkably similar to other structures from a subfamily of 
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-hydrolaseenzymes that includes pyrazinamidase and N-carbamoylsarcosine amidohydrolase. However, PhzD catalyzesunrelated chemistry and lacks a nucleophilic cysteine found in its close structural relatives. The vinylether hydrolysis catalyzed by PhzD represents yet another example of the catalytic diversity seen in the/-hydrolase family, whose members are also known to hydrolyze amides, phosphates, phosphonates,epoxides, and C-X bonds.
/-hydrolaseenzymes that includes pyrazinamidase and N-carbamoylsarcosine amidohydrolase. However, PhzD catalyzesunrelated chemistry and lacks a nucleophilic cysteine found in its close structural relatives. The vinylether hydrolysis catalyzed by PhzD represents yet another example of the catalytic diversity seen in the/-hydrolase family, whose members are also known to hydrolyze amides, phosphates, phosphonates,epoxides, and C-X bonds.