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. 2017 Mar 17;12(3):643-647.
doi: 10.1021/acschembio.7b00031. Epub 2017 Feb 15.

Substrate Trapping in the Siderophore Tailoring Enzyme PvdQ

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Free PMC article

Substrate Trapping in the Siderophore Tailoring Enzyme PvdQ

Kenneth D Clevenger et al. ACS Chem Biol. .
Free PMC article

Abstract

Siderophore biosynthesis by Pseudomonas aeruginosa enhances virulence and represents an attractive drug target. PvdQ functions in the type-1 pyoverdine biosynthetic pathway by removing a myristoyl anchor from a pyoverdine precursor, allowing eventual release from the periplasm. A circularly permuted version of PvdQ bypasses the self-processing step of this Ntn-hydrolase and retains the activity, selectivity, and structure of wild-type PvdQ, as revealed by a 1.8 Å resolution X-ray crystal structure. A 2.55 Å resolution structure of the inactive S1A/N269D-cpPvdQ mutant in complex with the pyoverdine precursor PVDIq reveals a specific binding pocket for the d-Tyr of this modified peptide substrate. To our knowledge, this structure is the first of a pyoverdine precursor peptide bound to a biosynthetic enzyme. Details of the observed binding interactions have implications for control of pyoverdine biosynthesis and inform future drug design efforts.

Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
PvdQ function, self-processing, and circular permutation. (A) Scheme shows PvdQ catalyzed hydrolysis to remove a myristoyl group from the biosynthetic precursor PVDIq to produce the ferribactin product that is eventually converted to pyoverdine (PVDI). The myristoyl group is shown in red, linker in orange, chromophore (precursors) in green, linear peptide portion in blue, and cyclic peptide portion in purple. (B) Self-proteolysis of the PvdQ proprotein leads to loss of a 23 residue linker peptide and formation of a heterodimer that relies on the newly formed β N-terminus to activate the catalytic nucleophile. (C) cpPvdQ has rearranged domains that circumvent the need for self-proteolysis.
Figure 2
Figure 2
X-ray crystal structure of PVDIq:S1A/N269D-cpPvdQ. (A) A cut-away view shown of S1A/N269D-cpPvdQ (gray) bound to PVDIq (blue) is shown with surfaces for each component, and the interacting P296–L301 α-helix (purple) shown as a ribbon. (B) A simulated annealing omit map (Fo-Fc) of bound PVDIq is shown with a density at 2.4 σ. A portion of unhydrolyzed PVDIq, shown as ball and stick, fits within the observed density. (C) Details of the binding pocket for the D-Tyr of PVDIq are shown. cpPvdQ (green) is shown with a portion of the P296–L301 α-helix as a ribbon. PVDIq (purple) is shown with H-bond distances in gray dotted lines and estimated cation–π and edge-to-face ππ interaction distances in green. Distances are subject to coordinate errors listed in Table S1.
Figure 3
Figure 3
PVDIq fragment ions confirming the structure of PVDIq. A map of b and y ions detected from PVDIq MS2 fragmentation is shown. The molecular cation of PVDIq with m/z = 1561.858 was detected in MS1 mode with a predicted molecular formula of C70H116N18O22 (1560.852 Da observed, 1560.851 Da theoretical, 0.2 ppm error). Diagnostic b and y ions characteristic of the expected fragmentation of PVDIq amide bonds in MS2 mode confirmed the ligand’s structure as that previously proposed for PVDIq (Figure 1). All fragments ions are within 5 ppm of theoretical m/z values and match those previously reported for PVDIq.

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