Pseudomonas aeruginosa pyoverdine maturation enzyme PvdP has a noncanonical domain architecture and affords insight into a new subclass of tyrosinases

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.

Keywords: Pseudomonas aeruginosa (P. aeruginosa); biosynthesis; copper; enzyme; infectious disease; protein structure; siderophore; streptavidin; tyrosinase.

Figure 1.

A, overview of PVD biosynthesis from fatty acids (FA) and proteinogenic and nonproteinogenic amino acids. l-Asp-SA, l-Asp-semialdehyde; l-Dab, l-2,4-diaminobutyrate; l-Orn, l-ornithine; l-OH-Orn, l-N⁵-hydroxy-Orn; l-fOH-Orn, l-N⁵-formyl-N⁵-hydroxyornithine. The precursor of PVD assembles in the cytosol, undergoes maturation in the periplasm, and binds ferric ion outside of the cell. B, current understanding of chromophore formation in PVD biosynthesis from ferribactin (left). TE, thioesterase domain. PvdP and the tyrosyl moiety of PVD are highlighted in red.

Figure 2.

A, overall structure of the PvdP dimer in the apo form. The N-terminal β-barrel domain is shown in yellow, the C-terminal tyrosinase domain is in blue. The typical four-helix bundle of type-3 copper proteins is shown in light blue, the C-terminal section that gets displaced in the l-tyrosine complex is in red. Histidines of the CuA and CuB sites and the placeholder residue Tyr⁵³¹ are shown as sticks. Orange spheres indicate the expected positions of two Cu²⁺ ions deducted from the coordinates of Zn²⁺ in the l-tyrosine complex. B, topology diagram of the PvdP monomer. Helix α19 contains the placeholder residue Tyr⁵³¹ and becomes disordered in the complex with l-tyrosine. The topology diagram was drawn with TopDraw (57), and all molecular representations were prepared with PyMOL (51).

Figure 3.

The N-terminal β-barrel domain of PvdP (PvdP-BBD) has structural similarity to streptavidin. A, two perpendicular views of PvdP-BBD. Access to the inside of the β-barrel is blocked by helix α1 and loop L1. The inside of the barrel is lined by the indicated aromatic residues. Removal of the side chain of Trp¹²⁸ would generate a cavity with a diameter of almost 10 Å. B, biotin-bound streptavidin from S. avidinii (PDB entry code 3RY2; Ref. 23) shown from similar orientations as PvdP-BBD. Note that the hypothetical biotin-binding site in PvdP-BBD is blocked.

Figure 4.

Sequence alignment of the CuA and CuB sites of PvdP with other type-3 copper proteins of the α-subclass (HcOd, hemocyanin A-type, Octopus dofleini; AuSCg, plant aurone synthase, Coreopsis grandiflora; TyrSc, tyrosinase, S. castaneoglobisporus; TyrBm, tyrosinase, B. megaterium) and β-subclass (HCPi, hemocyanin A, Panulirus interruptus; HcLp, hemocyanin II, L. polyphemus; TyrDm, tyrosinase, D. melanogaster) as defined previously (31). Copper-coordinating histidines are shown in red, and similar residues are in blue (α-subclass only), gray (β-subclass only), or yellow (both α- and β-subclasses). Strictly conserved residues are shown in bold. Stars highlight two phenylalanines found in all type-3 copper proteins (Phe²⁶⁷ and Phe⁴²⁸ in PvdP). Filled circles mark every 10th amino acid of the top sequence.

Figure 5.

Details of the tyrosinase active site of PvdP. A, the TYD of PvdP contains a CuA and a CuB site, which were loaded with Zn²⁺ (gray spheres) in the complex with l-tyrosine determined here (thin black lines). In the apo structure, the placeholder residue Tyr⁵³¹ occupies the binding site of the substrate's tyrosyl moiety, but autoxidation is hindered by holding the residue further away from the metal atoms (interaction with Gly⁴¹⁷). Glu³⁷¹ and Asp³⁷⁶ bind a water molecule that is implied in substrate deprotonation in other tyrosinases. Met²⁷⁰ and Met²⁷⁴ shield the active site from the solvent and could play a role in loading the enzyme with Cu²⁺. B, two representations of the molecular surface of PvdP in complex with l-tyrosine. The left side shows an electrostatic surface at ±10 k_BT/e; the surface on the right has been colored according to the two chains of the PvdP homodimer. C, closeup of the l-tyrosine–binding site. Electrostatic potentials were calculated with APBS (46); A and C are cross-eyed stereoplots.