Abstract
Pyoverdines (PVDs) are important chromophore-containing siderophores of fluorescent pseudomonad bacteria such as the opportunistic human pathogen Pseudomonas aeruginosa in which they play an essential role in host infection. PVD biosynthesis encompasses a complex pathway comprising cytosolic nonribosomal peptide synthetases that produce a polypeptide precursor that periplasmic enzymes convert to the final product. The structures of most enzymes involved in PVD chromophore maturation have been elucidated, but the structure of the essential tyrosinase PvdP, a monooxygenase required for the penultimate step in PVD biosynthesis, is not known. Here, we closed this gap by determining the crystal structure of PvdP in an apo and tyrosine-complexed state at 2.1 and 2.7 Å, respectively. These structures revealed that PvdP is a homodimer, with each chain consisting of a C-terminal tyrosinase domain and an N-terminal eight-stranded β-barrel reminiscent of streptavidin that appears to have a structural role only. We observed that ligand binding leads to the displacement of a "placeholder" tyrosine that blocks the active site in the apo structure. This exposes a large, deep binding site that seems suitable for accommodating ferribactin, a substrate of PvdP in PVD biosynthesis. The binding site consists almost exclusively of residues from the tyrosinase domain. Of note, we also found that this domain is more closely related to tyrosinases from arthropods rather than to tyrosinases from other bacteria. In conclusion, our work unravels the structural basis of PvdP's activity in PVD biosynthesis, observations that may inform structure-guided development of PvdP-specific inhibitors to manage P. aeruginosa infections.
Highlights
Pyoverdines (PVDs) are important chromophore-containing siderophores of fluorescent pseudomonad bacteria such as the opportunistic human pathogen Pseudomonas aeruginosa in which they play an essential role in host infection
The structures of most enzymes involved in PVD chromophore maturation have been elucidated, but the structure of the essential tyrosinase PvdP, a monooxygenase required for the penultimate step in PVD biosynthesis, is not known
Because PvdP is essential for PVD biosynthesis [11] and was found to be up-regulated in an acute burned mouse P. aeruginosa infection model [21], it may be a suitable target for anti-infectives, but the lack of high-resolution structures hampers the development of such inhibitors
Summary
The biosynthesis of the fluorescent dihydroxyquinoline moiety involves an oxidative cascade [20] in which the tyrosinase PvdP acts as a cresolase (monooxidase) to form a catechol from the D-Tyr moiety of the PVD precursor ferribactin first, followed by catecholase (oxidase) activity to create an o-quinone that undergoes intramolecular cyclization before PvdO performs a final oxidation to the fluorophore (Fig. 1B; Refs. 14 and 15). The identity between PvdP and other tyrosinases is low, a tyrosinase-typical type-3 dicopper center involving six essential, highly conserved histidines has been revealed by homology modeling. Based on these findings, it has been proposed that PvdP is the first member of a new tyrosinase family [14]. Comparison of the apo structure with a ligand complex shows that the active site is blocked by a “placeholder” residue (Tyr531) in the apo form, which is typical for tyrosinases. Ligand binding displaces this placeholder and exposes an extensive binding site to host the large ferribactin substrate. The binding site consists mainly of residues from the tyrosinase domain and is lined by a small proportion of the N terminus of the second monomer, suggesting that the streptavidin-like domain primarily possesses a structural rather than a functional role
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