Crystal Structure of the Pyocyanin Biosynthetic Protein PhzS

Abstract

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-1-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 A. 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.

The Pseudomonas aeruginosa efflux pump MexGHI-OpmD transports a natural phenazine that controls gene expression and biofilm development

“…1E). We found that the deletion of mex leads to delayed wrinkling solely in strains capable of producing 5-Me-PCA (i.e., strains that contain the phenazine biosynthetic enzymes PhzA-G and PhzM) (43,44).…”

“…5-Me-PCA, a reactive compound with a higher redox potential than the well-studied P. aeruginosa phenazines, has not been fully purified from wild-type P. aeruginosa cultures, although various observations indicate that it is the intermediate between phenazine-1-carboxylate (PCA) and pyocyanin in the phenazine biosynthetic pathway (38)(39)(40). Among these are the facts that the red phenazine aeruginosin A (5-methyl-7-amino-1-carboxyphenazinium betaine) is overproduced by P. aeruginosa phzS mutants (41,42), which lack the hydroxylase required to convert 5-Me-PCA to pyocyanin (43,44), and that E. coli expressing phzM converts PCA into a red pigment (42). Interestingly, the gene required for production of 5-Me-PCA from PCA, phzM, is located next to the mex operon on the P. aeruginosa chromosome (Fig.…”

Pseudomonas aeruginosa - Candida albicans Interactions: Localization and Fungal Toxicity of a Phenazine Derivative

“…1A) (45). While both its precursor, PCA, and its derivative, pyocyanin, are detected at near millimolar concentrations in culture supernatants, the PhzM intermediate, proposed to be 5MPCA, has not been detected in supernatants and has been proposed to be unstable (4,13,39). In P. aeruginosa, phenazine production is controlled by a number of regulators, including those involved in cell density-dependent signaling, referred to as quorum sensing.…”

Delineation of the Caffeine C-8 Oxidation Pathway in Pseudomonas sp. Strain CBB1 via Characterization of a New Trimethyluric Acid Monooxygenase and Genes Involved in Trimethyluric Acid Metabolism

“…The supernatant was then analyzed by an HPLC system equipped with a Shimadzu LCMS-2010EV single-stage quadruple mass analyzer. A parallel control reaction was carried out with 16 O 2 (air), and the product was analyzed. Liquid chromatography-mass spectrometry (LC-MS) analysis of time course of product formation.…”

“…Homology modeling. A BLASTP search with TMU-oxidizing enzyme and HpxO (30) as queries against the PDB identified a flavin-containing hydroxylase involved in pyocyanin biosynthesis (PhzS) from Pseudomonas aeruginosa (16) as the best match, with 30% sequence identity to both. PhzS belongs to the family of flavin-dependent aromatic hydroxylases, of which the best characterized is p-hydroxybenzonate hydroxylase (PHBH).…”