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Identification of inhibitors of PvdQ, an enzyme involved in the synthesis of the siderophore pyoverdine.

Wurst JM, Drake EJ, Theriault JR, Jewett IT, VerPlank L, Perez JR, Dandapani S, Palmer M, Moskowitz SM, Schreiber SL, Munoz B, Gulick AM - ACS Chem. Biol. (2014)

Bottom Line: Here, we describe the discovery of ML318, a biaryl nitrile inhibitor of PvdQ acylase.ML318 inhibits PvdQ in vitro (IC50 = 20 nM) by binding in the acyl-binding site, as confirmed by the X-ray crystal structure of PvdQ bound to ML318.Additionally, the PvdQ inhibitor is active in a whole cell assay, preventing pyoverdine production and limiting the growth of P. aeruginosa under iron-limiting conditions.

View Article: PubMed Central - PubMed

Affiliation: The Broad Institute , Cambridge, Massachusetts 02142, United States.

ABSTRACT
Pseudomonas aeruginosa produces the peptide siderophore pyoverdine, which is used to acquire essential Fe(3+) ions from the environment. PvdQ, an Ntn hydrolase, is required for the biosynthesis of pyoverdine. PvdQ knockout strains are not infectious in model systems, suggesting that disruption of siderophore production via PvdQ inhibition could be exploited as a target for novel antibacterial agents, by preventing cells from acquiring iron in the low iron environments of most biological settings. We have previously described a high-throughput screen to identify inhibitors of PvdQ that identified inhibitors with IC50 values of ∼100 μM. Here, we describe the discovery of ML318, a biaryl nitrile inhibitor of PvdQ acylase. ML318 inhibits PvdQ in vitro (IC50 = 20 nM) by binding in the acyl-binding site, as confirmed by the X-ray crystal structure of PvdQ bound to ML318. Additionally, the PvdQ inhibitor is active in a whole cell assay, preventing pyoverdine production and limiting the growth of P. aeruginosa under iron-limiting conditions.

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Structure ofpyoverdine and the role of PvdQ in biosynthesis. (A)The mature pyoverdine siderophore is an undecapeptide containing anN-terminal sidearm, the chromophore, and a species-specific peptidechain. (B) PvdQ catalyzes the removal of the myristoyl group fromthe pyoverdine precursor.
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fig1: Structure ofpyoverdine and the role of PvdQ in biosynthesis. (A)The mature pyoverdine siderophore is an undecapeptide containing anN-terminal sidearm, the chromophore, and a species-specific peptidechain. (B) PvdQ catalyzes the removal of the myristoyl group fromthe pyoverdine precursor.

Mentions: Pyoverdine is composed of a conserved dihydroquinoline-type chromophoreand a peptide tail that varies among different Pseudomonas species (Figure 1A).11,21 Additionally, most strains produce variable pyoverdine isoformswith N-terminal succinate, succinamide, or glutamate moieties boundto the chromophore. PvdL, the first NRPS protein of the pyoverdinepathway, is shared among all sequenced pseudomonads and generatesthe peptide backbone that is converted into this chromophore.21 Interestingly, PvdL contains a N-terminal modulewith homology to fatty acyl-CoA ligases.23 We recently24 showed that this unusualNRPS architecture incorporates a myristate molecule, subsequentlyidentified as either myristic or myristoleic acid,25 at the N-terminus of an intermediate in pyoverdine biosynthesis.Additionally, we demonstrated that the incorporated fatty acid, whichis not present on mature pyoverdine, is removed by PvdQ,24 one of the 10 auxiliary proteins necessary forpyoverdine synthesis (Figure 1B).10 PvdQ belongs to a family of N-terminal nucleophile(Ntn) hydrolases that catalyze the cleavage of amide bonds via anacylated enzyme intermediate.26 PvdQ exhibitspromiscuity in activity and also cleaves acyl-homoserine lactonesthat are involved in quorum signaling.27,28


Identification of inhibitors of PvdQ, an enzyme involved in the synthesis of the siderophore pyoverdine.

Wurst JM, Drake EJ, Theriault JR, Jewett IT, VerPlank L, Perez JR, Dandapani S, Palmer M, Moskowitz SM, Schreiber SL, Munoz B, Gulick AM - ACS Chem. Biol. (2014)

Structure ofpyoverdine and the role of PvdQ in biosynthesis. (A)The mature pyoverdine siderophore is an undecapeptide containing anN-terminal sidearm, the chromophore, and a species-specific peptidechain. (B) PvdQ catalyzes the removal of the myristoyl group fromthe pyoverdine precursor.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4215858&req=5

fig1: Structure ofpyoverdine and the role of PvdQ in biosynthesis. (A)The mature pyoverdine siderophore is an undecapeptide containing anN-terminal sidearm, the chromophore, and a species-specific peptidechain. (B) PvdQ catalyzes the removal of the myristoyl group fromthe pyoverdine precursor.
Mentions: Pyoverdine is composed of a conserved dihydroquinoline-type chromophoreand a peptide tail that varies among different Pseudomonas species (Figure 1A).11,21 Additionally, most strains produce variable pyoverdine isoformswith N-terminal succinate, succinamide, or glutamate moieties boundto the chromophore. PvdL, the first NRPS protein of the pyoverdinepathway, is shared among all sequenced pseudomonads and generatesthe peptide backbone that is converted into this chromophore.21 Interestingly, PvdL contains a N-terminal modulewith homology to fatty acyl-CoA ligases.23 We recently24 showed that this unusualNRPS architecture incorporates a myristate molecule, subsequentlyidentified as either myristic or myristoleic acid,25 at the N-terminus of an intermediate in pyoverdine biosynthesis.Additionally, we demonstrated that the incorporated fatty acid, whichis not present on mature pyoverdine, is removed by PvdQ,24 one of the 10 auxiliary proteins necessary forpyoverdine synthesis (Figure 1B).10 PvdQ belongs to a family of N-terminal nucleophile(Ntn) hydrolases that catalyze the cleavage of amide bonds via anacylated enzyme intermediate.26 PvdQ exhibitspromiscuity in activity and also cleaves acyl-homoserine lactonesthat are involved in quorum signaling.27,28

Bottom Line: Here, we describe the discovery of ML318, a biaryl nitrile inhibitor of PvdQ acylase.ML318 inhibits PvdQ in vitro (IC50 = 20 nM) by binding in the acyl-binding site, as confirmed by the X-ray crystal structure of PvdQ bound to ML318.Additionally, the PvdQ inhibitor is active in a whole cell assay, preventing pyoverdine production and limiting the growth of P. aeruginosa under iron-limiting conditions.

View Article: PubMed Central - PubMed

Affiliation: The Broad Institute , Cambridge, Massachusetts 02142, United States.

ABSTRACT
Pseudomonas aeruginosa produces the peptide siderophore pyoverdine, which is used to acquire essential Fe(3+) ions from the environment. PvdQ, an Ntn hydrolase, is required for the biosynthesis of pyoverdine. PvdQ knockout strains are not infectious in model systems, suggesting that disruption of siderophore production via PvdQ inhibition could be exploited as a target for novel antibacterial agents, by preventing cells from acquiring iron in the low iron environments of most biological settings. We have previously described a high-throughput screen to identify inhibitors of PvdQ that identified inhibitors with IC50 values of ∼100 μM. Here, we describe the discovery of ML318, a biaryl nitrile inhibitor of PvdQ acylase. ML318 inhibits PvdQ in vitro (IC50 = 20 nM) by binding in the acyl-binding site, as confirmed by the X-ray crystal structure of PvdQ bound to ML318. Additionally, the PvdQ inhibitor is active in a whole cell assay, preventing pyoverdine production and limiting the growth of P. aeruginosa under iron-limiting conditions.

Show MeSH
Related in: MedlinePlus