Structure and Mechanism of Pseudomonas aeruginosa PhzD, an Isochorismatase from the Phenazine Biosynthetic Pathway^,,

Abstract

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 A crystal structure of native PhzD, and the 1.6 A 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.

A study of noncovalent protein complexes by matrix-assisted laser desorption/ionization

“…The crystal structure has been solved and it is a dimer in the solid state. The solution molecular mass of this enzyme was determined by a combination of light scattering and interferometric refractometry [ 16] and it is also a dimer in solution. The MALDI spectrum of this enzyme (Figure 1), using sample preparation described here, showed a major peak of homodimer at m/z 50K and a major peak at 25K of monomer with minor peaks from other high-order oligomers such as trimer and tetramer.…”

The YebC Family Protein PA0964 Negatively Regulates thePseudomonas aeruginosaQuinolone Signal System and Pyocyanin Production

“…The expression of phzA1 accounts for the majority of phenazine production (6,49). In addition, the gene products of phzM, phzH, and phzS are required to convert the intermediate phenazine-1-carboxylic acid to other final products, including phenazine-1-carboxamide, 1-hydroxphenazine, and pyocyanin (33,34,38). Production of phenazine compounds is tightly regulated by quorum-sensing systems in P. aeruginosa.…”

A Novel Enzyme Conferring Streptothricin Resistance Alters the Toxicity of Streptothricin D from Broad-spectrum to Bacteria-specific

“…Isochorismatase (EC 3.3.2.1) is an enzyme (EntB) occurring in E. coli and is produced during the biosynthesis of enterobactin, an iron-chelating product derived from chorismic acid and involved in the transport of iron from the bacterial environment into the cell cytoplasm (30 -32). The phzD gene encoding an isochorismatase-related enzyme, which shares 46% identity with the EntB isochorismatase, is also known to participate in phenazine biosynthesis in Pseudomonas strains (33). Parsons et al (33) have recently reported that the structure of PhzD is remarkably similar to other structures from a subfamily of ␣/␤-hydrolase-fold enzymes, whose members are known to hydrolyze amides, phosphates, phosphonates, epoxides, and C-X bonds.…”