Rational Design of a Transition State Analogue with Picomolar Affinity for Pseudomonas aeruginosa PvdQ, a Siderophore Biosynthetic Enzyme

• Published in: ACS chemical biology 2013-10, Vol.8 (10), p.2192-2200
• Main Authors: Clevenger, Kenneth D, Wu, Rui, Er, Joyce A. V, Liu, Dali, Fast, Walter
• Format: Article
• Language: English
• Subjects: Biological Assay; Chemistry, Pharmaceutical; Crystallography, X-Ray; Drug Design; Enzyme Activation - drug effects; Enzyme Inhibitors - chemistry; Enzyme Inhibitors - pharmacology; Inhibitory Concentration 50; Ligands; Models, Molecular; Pseudomonas aeruginosa - drug effects; Pseudomonas aeruginosa - enzymology; Siderophores - chemistry; Siderophores - metabolism; Substrate Specificity
• ISSN: 1554-8929; 1554-8937
• DOI: 10.1021/cb400345h

The Pseudomonas aeruginosa enzyme PvdQ can process different substrates involved in quorum-sensing or in siderophore biosynthesis. Substrate selectivity was evaluated using steady-state kinetic constants for hydrolysis of N-acyl-homoserine lactones (HSLs) and p-nitrophenyl fatty acid esters. PvdQ prefers substrates with alkyl chains between 12 and 14 carbons long that do not bear a 3-oxo substitution and is revealed here to have a relatively high specificity constant for selected N-acyl-HSLs (k cat/K M = 105 to 106 M–1 s–1). However, endogenous P. aeruginosa N-acyl-HSLs are ≥100-fold disfavored, supporting the conclusion that PvdQ was not primarily evolved to regulate endogenous quorum-sensing. PvdQ plays an essential biosynthetic role for the siderophore pyoverdine, on which P. aeruginosa depends for growth in iron-limited environments. A series of alkylboronate inhibitors was found to be reversible, competitive, and extremely potent (K i ≥ 190 pM). A 1.8 Å X-ray structure shows that 1-tridecylboronic acid forms a monocovalent bond with the N-terminal β-chain Ser residue in the PvdQ heterodimer, mimicking a reaction transition state. This boronic acid inhibits growth of P. aeruginosa in iron-limited media, reproducing the phenotype of a genetic pvdQ disruption, although co-administration of an efflux pump inhibitor is required to maintain growth inhibition. These findings support the strategy of designing boron-based inhibitors of siderophore biosynthetic enzymes to control P. aeruginosa infections.