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

Priya Philem,Iain L Lamont,Sigurd M Wilbanks,Bill C Hawkins,Torsten Kleffmann,Sinan Gai

doi:10.3390/ijms22042211

Priya Philem, Iain L Lamont + Show 4 more

Open Access

https://doi.org/10.3390/ijms22042211

Copy DOI

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.

Highlights

IntroductionPyoverdines are yellow green fluorescent siderophores secreted by Pseudomonads, including the opportunistic pathogen P. aeruginosa
Siderophores are low molecular weight (200–2000 Da) ferric ion-specific chelating agents secreted by bacteria and fungi in order to obtain iron required for growth [1].Pyoverdines are yellow green fluorescent siderophores secreted by Pseudomonads, including the opportunistic pathogen P. aeruginosa
As well as confirming that this enzyme catalyses the synthesis of fOHOrn from OHOrn and fTHF, our results demonstrate the importance of likely active site residues in catalysis and indicate that PvdF is likely to be part of the siderosome multienzyme complex that carries out pyoverdine synthesis

Summary

Introduction

Pyoverdines are yellow green fluorescent siderophores secreted by Pseudomonads, including the opportunistic pathogen P. aeruginosa. Pyoverdines consist of a 2,3-diamino6,7-dihydroxyquinoline fluorophore that gives yellow green fluorescence, a variable acyl side chain attached to the 3-amino group of the fluorophore, and a strain-specific peptide backbone of 6–12 amino acids (Figure 1), including D-isomers and other non-proteinaceous amino acids [7,8]. One of these is N5 -L-formylhydroxyornithine (fOHOrn), present in the pyoverdine of P. aeruginosa reference strain PAO1.

Objectives

Methods

Results

Conclusion