Investigation and engineering of PvdQ, a Pseudomonas aeruginosa enzyme at the nexus of quorum sensing and iron uptake pathways

Abstract

The gram-negative human pathogen Pseudomonas aeruginosa is a widespread global health concern. Two key pathways of P. aeruginosa that are involved in its infection and/or survival within mammalian hosts are the pyoverdine biosynthetic pathway, which is necessary for iron acquisition, and the N-acyl-homoserine lactone (AHL) quorum sensing (QS) system, which is necessary for P. aeruginosa virulence and infection. The P. aeruginosa enzyme PvdQ is unique in that it is proposed to play roles in both these pathways. To investigate the role of PvdQ in P. aeruginosa, the enzyme’s in vitro substrate profile was characterized and found to favor substrates with unsubstituted myristic acyl groups, supporting a role for PvdQ in pyoverdine biosynthesis, but challenging its proposed role in regulating P. aeruginosa QS. Knowledge of PvdQ’s substrate preference and mechanism enabled rational inhibitor design, leading to the discovery of long chain (≥ 10 carbons) n-alkylboronic acids as highly potent transition state analog inhibitors of PvdQ, with 1-tridecylboronic acid having a Ki of 200 ± 40 pM and having the ability to recapitulate the growth phenotype of a pvdQ transposon insertion strain of P. aeruginosa under certain experimental conditions. Investigation of PvdQ inhibition by alkylboronic acids was extended to short- and medium-chain alkylboronic acids and revealed an eight order of magnitude span of affinity that is linearly related to alkyl chain length. Through structural studies, short-chain alkylboronic acids (≤ 4 carbons) were found to adopt an alternate binding conformation relative to other alkylboronic acids, which suggests a mechanism by which PvdQ excludes short substrates such as P. aeruginosa QS molecule N-butyroyl-homoserine lactone. Finally, a circular permutation of PvdQ (cpPvdQ) was designed and constructed in order to uncouple the catalytic and self-processing activities of PvdQ and to facilitate the production of mutants that affect catalysis and specificity. The cpPvdQ monomer recapitulates the catalytic and structural features of PvdQ. A catalytically impaired cpPvdQ mutant that is shown to maintain affinity for PvdQ’s native substrate, the pyoverdine precursor, but does not hydrolyze the substrate, has been produced and will be a valuable tool for future efforts to trap a PvdQ derivative in complex with the pyoverdine precursor.