Identification of Active Site Residues of the Siderophore Synthesis Enzyme PvdF and Evidence for Interaction of PvdF with a Substrate-Providing Enzyme

Abstract

The problematic opportunistic pathogen Pseudomonas aeruginosa secretes a siderophore, pyoverdine. Pyoverdine scavenges iron needed by the bacteria for growth and for pathogenicity in a range of different infection models. PvdF, a hydroxyornithine transformylase enzyme, is essential for pyoverdine synthesis, catalysing synthesis of formylhydroxyornithine (fOHOrn) that forms part of the pyoverdine molecule and provides iron-chelating hydroxamate ligands. Using a mass spectrometry assay, we confirm that purified PvdF catalyses synthesis of fOHOrn from hydroxyornithine and formyltetrahydrofolate substrates. Site directed mutagenesis was carried out to investigate amino acid residues predicted to be required for enzymatic activity. Enzyme variants were assayed for activity in vitro and also in vivo, through measuring their ability to restore pyoverdine production to a pvdF mutant strain. Variants at two putative catalytic residues N168 and H170 greatly reduced enzymatic activity in vivo though did not abolish activity in vitro. Change of a third residue D229 abolished activity both in vivo and in vitro. A change predicted to block entry of N10-formyltetrahydrofolate (fTHF) to the active site also abolished activity both in vitro and in vivo. A co-purification assay showed that PvdF binds to an enzyme PvdA that catalyses synthesis of hydroxyornithine, with this interaction likely to increase the efficiency of fOHOrn synthesis. Our findings advance understanding of how P. aeruginosa synthesises pyoverdine, a key factor in host–pathogen interactions.

Novel Insights on Pyoverdine: From Biosynthesis to Biotechnological Application

“…Interestingly, Gasser and colleagues [ 63 ] showed, using the FRET–FLIM (Förster resonance energy transfer measured by fluorescence lifetime microscopy) technique, that PvdA interacts physically with PvdJ, PvdI, PvdL, and PvD, suggesting the presence of a strongly organized multienzymatic complex coordinating the PVD biosynthesis. Similarly, Philem and collaborators [ 64 ] demonstrated the fundamental role played by PvdF through a mutagenesis experiment, as the mutants carrying the inactivated enzyme had a reduced production of PVDI. Therefore, the authors suggest a possible switch to regulate PVD production for biotechnological purposes.…”

“…As just mentioned, two other NRPSs directly involved in the elongation of the peptide are PvdJ and PvdD, catalyzing the addition of L-Lys, L-hfOrn, and two L-Thr residues [ 63 , 64 , 65 ]. The activity of two auxiliary enzymes, PvdG and MbtH, appears to be required for the intra-cytoplasmic maturation of the ferribactin.…”

New Insights into the Role of Metals in Host–Pathogen Interactions

“…Two other contributions to this Special Issue concern iron acquisition via siderophores in P. aeruginosa . The paper by Philem et al [ 8 ] investigated the role of PvdF, a hydroxyornithine transformylase enzyme, in the synthesis of pyoverdine, the siderophore with the highest affinity for iron among those synthesized by this opportunistic pathogen. Through a mass spectrometry assay, their study confirmed the predicted activity of the enzyme.…”