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 spec-trometry assay, we confirm that purified PvdF catalyses synthesis of fOHOrn from hydroxyorni-thine 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 hy-droxyornithine, 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–path-ogen interactions.