Structural Characterization and High-Throughput Screening of Inhibitors of PvdQ, an NTN Hydrolase Involved in Pyoverdine Synthesis.

Pseudomonas aeruginosa is an opportunistic gram-negative pathogen that causes nosocomial infections and chronic lung infections in cystic fibrosis patients ((1), (2)) These infections are established in the form of a biofilm that is relatively insensitive to immune responses and antibiotics (3). This native resistance and persistent infection in the face of current antibacterial drugs has far reaching consequences for patient morbidity and mortality and also demonstrates a need to identify new strategies and therapies to combat this pathogen. Targeting novel essential bacterial pathways that are responsible for the acquisition of essential nutrients is one possible mechanism for development of new anti-infective agents (4). Iron is a necessary trace element for nearly all living organisms and plays key catalytic and structural roles in proteins (5). Despite its relative abundance, free iron (Fe3+) acquisition poses a challenge to bacteria due to toxicity and poor solubility. As a result, bacteria have evolved synthetic pathways to produce and secrete high affinity sequestering agents called siderophores that bind to iron and are actively transported back into the cell (6). In many bacteria, specialized peptide siderophores are produced by modular enzymes known as non-ribosomal peptide synthetases (NRPSs). These enzymes are molecular assembly lines, organized with multiple catalytic domains joined in a single protein (7). To produce the siderophore compounds, many NRPS proteins work in concert with other NRPSs or accessory proteins. These supplementary enzymes are involved in the synthesis of building blocks, siderophore maturation and export, import of the Fe3+-siderophore complex, or the removal of Fe3+ from the imported siderophore (8). Pyoverdine is the primary iron siderophore produced by P. aeruginosa and has been associated with infection in multiple disease models (5). Multiple isoforms of pyoverdine have been identified in P. aeruginosa (Figure 1), all of which are composed of a cyclic peptide chain synthesized by the four large cytoplasmic NRPSs (PvdL, PvdI, PvdJ, PvdD), a chemically modified dihydroxyquinoline-based chromophore that is responsible for iron binding, and an N-terminal side chain bound to the chromophore ((9), (10)). Along with the NRPSs that produce the peptide chain, eleven other proteins have been identified that are critical to pyoverdine production (11). These proteins play a significant role in pyoverdine synthesis, including cyclization, export, and final maturation in the periplasmic space ((5), (12)). Several proteins are well characterized, including the ornithine hydroxylase PvdA (13), the aminotransferase PvdH (14), and the hydroxyornithine transformylase PvdF (15) Although the exact roles of many of the tailoring enzymes are not known, their involvement in this essential siderophore catalytic pathway presents them as attractive targets for new antibiotic development (16). Figure 1 The pyoverdine siderophore produced by the human pathogen Pseudomonas aeruginosa. Several isoforms have been identified that vary at the N-terminal residue. A potential strategy for identifying new treatments for P. aeruginosa infections is finding small molecules that interfere with maturation and expression of critical siderophores or quorum sensors, both of which have been implicated in biofilm formation ((17), (18)) and bacterial virulence (19). High throughput screening (HTS) methods could be used to identify compounds that disrupt the maturation processes in these metabolic pathyways, and indeed this method has already been proven effective in identifying potential small molecule inhibitors of bacterial signaling molecules (20). In this regard, we have investigated the fatty acylase PvdQ, a critical protein in pyoverdine synthesis. PvdQ belongs to the NTN hydrolase family (21), whose members are produced as inactive proteins and autoproteolytically cleaved to produce active α/β-heterodimers. The N-terminus of the β–chain is preferentially a serine that is responsible both for autoproteolysis and the catalytic cleavage of acyl esters (22). PvdQ was identified as a periplasmic quorum quenching protein that cleaves acyl homoserine lactones (23). The crystal structure has been reported in this context (21) for both native PvdQ and a covalently acylated enzyme. However, the location of PvdQ in the pyoverdine synthetic operon and recent reports suggest that its primary role is in pyoverdine maturation. Quax et al (24) have demonstrated that a pvdQ knockout shows multiple reduced virulence factors, including pyoverdine production, growth in iron-limiting media, swarming motility, and biofilm formation. These phenotypes could not be linked with the presence or absence of acylated signaling molecules. Additionally, an acyl-pyoverdine precursor termed PVDIq has recently been isolated from a pvdQ mutant, providing a direct link between PvdQ and maturation of the pyoverdine precursor (25). Herein, we describe the fatty acid adenylating activity of the initial module of PvdL, demonstrating the chemical nature of the acylated pyoverdine peptide precursor. The acyl pyoverdine is purified and shown through biochemical analysis to serve as a substrate for PvdQ. We additionally describe the development and validation of a high throughput screen for small molecule inhibitors of PvdQ. The assay exploits the promiscuous activity of PvdQ to produce colorimetric or fluorimetric by-products. Results from initial small library screenings are presented and validated by kinetic analyses. Structural studies are also presented showing PvdQ covalently acylated with the acyl moiety substrate derived from the acyl-pyoverdine precursor, as well as bound to two novel small molecule inhibitors identified from the library screen. These biochemical and structural studies demonstrate that the described assay is a viable method for identification of small molecule inhibitors that may prevent pyoverdine maturation and hinder cell growth under iron limiting conditions.