PvdF of pyoverdin biosynthesis is a structurally unique N10-formyltetrahydrofolate-dependent formyltransferase

Abstract

The hydroxyornithine transformylase from Pseudomonas aeruginosa is known by the gene name pvdF, and has been hypothesized to use N 10 -formyltetrahydrofolate (N 10 -fTHF) as a co-substrate formyl donor to convert N 5 -hydroxyornithine (OHOrn) to N 5 -formyl-N 5 -hydroxyornithine (fOHOrn). PvdF is in the biosynthetic pathway for pyoverdin biosynthesis, a siderophore generated under iron-limiting conditions that has been linked to virulence, quorum sensing and biofilm formation. The structure of PvdF was determined by X-ray crystallography to 2.3 Å, revealing a formyltransferase fold consistent with N 10 -formyltetrahydrofolate dependent enzymes, such as the glycinamide ribonucleotide transformylases, N-sugar transformylases and methionyl-tRNA transformylases. Whereas the core structure, including the catalytic triad, is conserved, PvdF has three insertions of 18 or more amino acids, which we hypothesize are key to binding the OHOrn substrate. Steady state kinetics revealed a non-hyperbolic rate curve, promoting the hypothesis that PvdF uses a random-sequential mechanism, and favors folate binding over OHOrn.

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

“…The recombinant proteins were purified using Ni 2+ affinity resin and size exclusion chromatography ( Figure S2 ). Soluble and homogenous proteins were obtained, with PvdF purifying as a monomer as also reported by others [ 11 ].…”

“…Reactions were carried out for up to 4 h and the highest amount of fOHOrn was present after 3 h of incubation ( Figure 2 E). A similar assay for PvdF was developed independently and reported during the course of this research [ 11 ]. Those researchers also found that purified PvdF catalyses the formation of fOHOrn from OHOrn and fTHF substrates.…”

“…PvdF, like other fTHF dependent enzymes, has two subdomains [ 11 ]. Thermal stability experiments revealed two protein melting events that are likely to correspond to independent melting of the two subdomains.…”

“…PvdA catalyses conversion of l -ornithine (Orn) to L-N 5 -hydroxyornithine (OHOrn) [ 9 ]. PvdF formylates OHOrn, with N 10 -formyltetrahydrofolate (fTHF) as the co-substrate, to produce fOHOrn ( Figure 1 ) [ 10 , 11 ]. fOHOrn is then incorporated into the pyoverdine precursor peptide by the non-ribosomal peptide synthetases (NRPSs) PvdI and PvdJ [ 12 , 13 , 14 ].…”

“…Therefore, no direct evidence exists for which residues participate in substrate binding and catalysis. The closest characterised structural and functional homologue of PvdF is glycinamide ribonucleotide transformylase (GART) from E. coli [ 10 , 11 ]. GART catalyses the formylation of N 1 -(5-phospho- d -ribosyl)glycinamide (GAR) with fTHF as the formyl donor as part of the de novo purine synthesis pathway.…”