PvdP Is a Tyrosinase That Drives Maturation of the PyoverdineChromophore in Pseudomonas aeruginosa

Pol Nadal-Jimenez,a,b Gudrun Koch,a,c Carlos R. Reis,a,d Remco Muntendam,a Hans Raj,a C. Margot Jeronimus-Stratingh,e

Robbert H. Cool,a Wim J. Quaxa

Department of Pharmaceutical Biology, University of Groningen, Antonius Deusinglaan, Groningen, The Netherlandsa; Instituto Gulbenkian de Ciência, Oeiras, Portugalb;Research Center for Infectious Diseases ZINF, Würzburg Universität, Josef-Schneider-Strasse 2, Würzburg, Germanyc; Department of Cell Biology, UT SouthwesternMedical Center, Dallas, Texas, USAd; Mass Spectrometry Core Facility, University of Groningen, A. Deusinglaan, Groningen, The Netherlandse

The iron binding siderophore pyoverdine constitutes a major adaptive factor contributing to both virulence and survival in fluo-rescent pseudomonads. For decades, pyoverdine production has allowed the identification and classification of fluorescent andnonfluorescent pseudomonads. Here, we demonstrate that PvdP, a periplasmic enzyme of previously unknown function, is atyrosinase required for the maturation of the pyoverdine chromophore in Pseudomonas aeruginosa. PvdP converts the nonfluo-rescent ferribactin, containing two iron binding groups, into a fluorescent pyoverdine, forming a strong hexadentate complexwith ferrous iron, by three consecutive oxidation steps. PvdP represents the first characterized member of a small family of ty-rosinases present in fluorescent pseudomonads that are required for siderophore maturation and are capable of acting on largepeptidic substrates.

Iron is an essential element used as a cofactor in a large number ofreactions in all living organisms. However, in the presence of

oxygen, iron occurs predominantly in the Fe3� state, which ischaracterized by a very low solubility, and thus it is also one of themajor growth-limiting factors. To overcome this problem, a largenumber of microorganisms produce siderophores, iron-chelatingmolecules that are secreted into the environment where theystrongly bind and facilitate the import of Fe3� inside the cells(1–7). Fluorescent pseudomonads, including the human patho-gen Pseudomonas aeruginosa and the plant pathogen Pseudomonassyringae, rely on pyoverdine (PVD) siderophores to perform thistask. PVDs are the major siderophores in fluorescent pseudomon-ads and consist of a variable short peptide chain (containing twoiron binding residues) linked to a conserved catecholate chro-mophore moiety (that provides a third iron binding site) (7).Three types of PVDs (PVDI, PVDII, and PVDIII) have been foundto be produced by different strains of P. aeruginosa (8). Hereby, wewill refer to PVDI when describing PVD type I, the most studied P.aeruginosa PVD type found in multiple strains, including the well-characterized PAO1 and PA14. The PVDI pathway is complex,comprising approximately 20 different proteins involved in itsregulation, synthesis, maturation, transport, and uptake (Fig. 1)(7). While the synthesis and regulation of PVDI have been welldocumented in the past years, its maturation process is not eluci-dated. PVDI maturation starts with the transport of a PVDI pre-cursor (PVDIq) from the cytoplasm to the periplasm of P. aerugi-nosa by the ABC transporter PvdE (9, 10). It was recentlydemonstrated that this precursor (PVDIq) is a C14-acylated ferri-bactin that once in the periplasm is cleaved by the acylase PvdQ,leading to the formation of ferribactin (11, 12). Ferribactin is ayellowish precursor of PVDI in which the cyclization of the thirdring in the catechol moiety has not been accomplished. This mol-ecule lacks the typical greenish coloration of PVDI and its thirdstable iron binding group. Little is known about the followingsteps in the maturation of ferribactin leading to PVDI. The scarcedata on additional enzymes such as PvdN, PvdM, PvdO, or PvdPinvolved in PVDI maturation in the periplasm originate from the

quantification of PVDI production in a set of mutants initiallyidentified as iron-responsive (13).

Here, we demonstrate that PvdP, a previously uncharacterizedperiplasmic protein of unknown function in the maturation ofPVDI, is capable of converting the precursor, ferribactin, into thegreen fluorescent PVDI. Mechanistically, PvdP appears to hy-droxylate the D-tyrosine moiety of the tetrahydropyrimidine ring,resulting in a catechol functionality, which is followed by the for-mation of a third ring in the chromophore, leading to the forma-tion of the final PVDI chromophore (Fig. 2). In a final reactionsequence, PvdP restores the catechol functionality and creates thethird iron binding site in PVDI, needed for the high affinity ofFe3� (14). Thus, PvdP catalyzes three steps in the maturation offerribactin into fluorescent PVDI.

MATERIALS AND METHODSBacterial strains, plasmids, and culture conditions. P. aeruginosa PAO1was used as a source for the pvdP gene, which was PCR amplified andcloned with a C-terminal His tag fusion in pET26b (Novagen) using stan-dard cloning methods. For PvdP production, Escherichia coli BL21(DE3)harboring plasmid pET26B-pvdP_H6 cultures were grown overnight inLB medium and 1 ml was added to 1 liter of 2� TY medium (15) (bothmedia supplemented with 50 mg/liter kanamycin), incubated at 30°Cwith shaking at 250 rpm, and induced with 1 mM isopropyl-�-D-thioga-lactopyranoside (IPTG) at an optical density at 600 nm (OD600) of 0.6,followed by further incubation for approximately 16 h at 30°C. PvdQ wasproduced in E. coli DH10B (Invitrogen) harboring plasmid pMCT-pvdQas previously described (16). For protein localization assays, pvdP_H6 was

Received 21 November 2013 Accepted 6 May 2014

Published ahead of print 9 May 2014

Address correspondence to Wim J. Quax, w.j.quax@rug.nl.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/JB.01376-13.

Copyright © 2014, American Society for Microbiology. All Rights Reserved.

doi:10.1128/JB.01376-13

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PCR amplified from pET26B-pvdP_H6 and cloned in the shuttle vectorpME6032 (17), obtaining the plasmid pME-pvdP_H6. This plasmid wasthen transformed in the E. coli HB2151 wild type (WT) and the E. coliHB2151 �tatC mutant (18).

PvdP purification. Induced E. coli BL21(DE3)/pET26B-pvdP_H6cells were resuspended in 3 ml of sonication buffer (50 mM Tris-HCl, 500mM NaCl, 10% glycerol [vol/vol], 20 mM imidazole, 14 mM �-mercap-toethanol, 1% Tween [pH 8]) per gram of wet cells, lysed by sonication,and centrifuged at high speed to remove cell debris (1 h, 18,000 rpm). Thesupernatant was filtered using a cellulose acetate filter (0.2-�m pore size)and subsequently loaded on a nickel-charged HiTrap HP (5 ml) column(GE Healthcare). After the column was washed with 3 column volumes ofwashing buffer (50 mM Tris-HCl, 500 mM NaCl, 10% glycerol [vol/vol],20 mM imidazole, 14 mM �-mercaptoethanol [pH 8]), elution was per-formed with a linear gradient from 20 to 500 mM imidazole in elutionbuffer (50 mM Tris-HCl, 500 mM NaCl, 10% glycerol [vol/vol], 500 mMimidazole, 14 mM �-mercaptoethanol [pH 8]). PvdP was eluted with

approximately 100 mM imidazole. The elution buffer was exchanged to20 mM Tris-HCl buffer, pH 8, using a desalting HiTrap HP (5-ml) col-umn (GE Healthcare) and concentrated to 5 mg/ml using a 20,000MWCO Vivaspin column (Sartorius Stedim Biotech S.A., France). Theprotein was aliquoted, snap-frozen in liquid nitrogen, and stored at�80°C till further use.

PvdP cell localization. (i) Cell fractionation. To determine the cellu-lar localization of PvdP, pvdP_H6 was amplified from pET26b-pvdP_H6and cloned in the shuttle vector pME6032 (17) to obtain the plasmidpME-pvdP_H6. This plasmid was electroporated into the E. coli HB2151WT and the E. coli HB2151 �tatC mutant (18). The two strains weregrown overnight in 2� TY of LB medium at 37°C and 250 rpm. Afterincubation, cells were harvested and resuspended in phosphate-bufferedsaline (PBS) to an OD580 of 1. A 1-ml aliquot of cells was pipetted to a newtube and centrifuged, and the pellet was washed 3 times with 1 ml of PBSto remove extracellular proteins. After the last washing step, the cells wereresuspended in 1 ml of ice-cold Tris-HCl buffer (100 mM Tris [pH 8])

FIG 1 Production and export of the siderophore PVDI. PVDI biosynthesis starts when the low concentration of iron abrogates the formation of the stableFe-ferric uptake regulator (FUR) complex which otherwise binds to the pvdS promoter, blocking the production of the extracytoplasmic function (ECF)-sigmafactor PvdS. Upon translation, the regulator, PvdS, binds to the promoter IS boxes of PVDI genes, initiating transcription and translation of PVDI proteins. Thenonribosomal peptide synthases (NRPS) PvdL (41), PvdI, PvdJ, and PvdF initiate the assembly of the precursor siderophore PVDIq, which starts with a C14 fattyacid (FA) being attached to L-Glu and finishes with the release of PVDIq from PvdD by the thioesterase module (TE) after the last L-Thr has been incorporated.Enzymes PvdH, PvdA, and PvdF are responsible for the synthesis of the nonnatural amino-acids L-diaminobutyrate (L-Dab) and L-formyl-OH-ornithine that areincorporated into PVDIq by the four NRPS proteins. PvdE transports PVDIq to the periplasm, where the myristoleic acid is removed by the acylase PvdQ priorto the maturation of the chromophore, generating the precursor ferribactin (FB). The role of the uncharacterized PvdM, PvdN, and PvdO proteins in thematuration of ferribactin remains unknown; one possibility is that at least one of these proteins transforms the L-glutamate into one of the three residues foundat this position in PVDI. Mature PVDI is then secreted to the surrounding environment by PvdRT-OpmQ and by an as-yet-unidentified transporter. Immatureprecursors, such as ferribactin (42) and PVDIq (this study), have also been found in the secreted fraction of fluorescent pseudomonads, indicating that theseprecursors can also be secreted into the extracellular medium (dashed lines). Once in the medium, PVDI binds iron, forming the PVDI-Fe complex, which canbe taken up by the cell, where further steps are conducted.

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containing 20% (wt/vol) sucrose. A total of 1 ml of a Tris-HCl buffer (100mM Tris [pH 8]) containing 5 mM EDTA was added to the tube togetherwith 10 �l of lysozyme (2 mg/ml). The cells were incubated for 30 min atroom temperature and centrifuged for 20 min at 10,000 rpm. The super-natant containing the periplasmic fraction was collected and precipitatedovernight using 5% (wt/vol) trichloroacetic acid (TCA). Subsequently,the remaining pellet was resuspended in 1 ml of an ice-cold Tris-HClbuffer and the cells were lysed by sonication (2 min, 50%). After sonica-tion, the cells were centrifuged for 1 h at 13,000 � g. The supernatantcontaining the cytoplasmic proteins was precipitated overnight using 5%(wt/vol) TCA.

(ii) PvdP detection. After the periplasmic and cytoplasmic fractionswere separated, 80 �g of total protein was added to a 10% SDS polyacryl-amide gel. The proteins were then transferred to a nitrocellulose mem-brane, blocked with 5% skim milk, and probed with anti-6�His tag an-tibody (Sigma) to detect the presence of PvdP_H6. A control with a GroELantibody (Sigma) was used to assess the proper separation of the periplas-mic and the cytoplasmic fractions.

Isolation of PVDI and PVDI precursors. (i) PVDI. PVDI was isolatedfrom 1-liter cell-free culture supernatants of P. aeruginosa PAO1 WTgrown at 30°C, 250 rpm, for 48 h in low-iron CAA medium (16). Aftercentrifugation, the medium was separated with H2O and methanol byusing a C18 solid-phase extraction (SPE) column (Varian Megabond ElutC18; Agilent) separating three fractions: unbound fraction, 50% metha-nol-eluting fraction, and 100% MetOH-eluting fraction. The 50% elutedsample containing PVDI was collected, air dried at room temperature,resuspended in 1 ml H2O, and stored at �20°C till further analysis.

(ii) PVDIq. PVDIq was isolated from 1-liter cell-free culture superna-tants of the P. aeruginosa PAO1 �pvdQ mutant (19) grown at 30°C, 250rpm, for 48 h in low-iron medium (CAA). After centrifugation, the me-dium was separated with H2O and methanol using a C18-SPE column, asin the case of PVDI, but the 100% methanol elution containing the more

hydrophobic PVDIq was collected. The sample was air dried at roomtemperature, resuspended in 1 ml of 100% methanol, and stored at �20°Ctill further analyses.

(iii) Ferribactin. Ferribactin was obtained after mixing the 1 ml meth-anol solution containing PVDIq with 50 ml of 50 mM Tris-HCl, pH 8.8,adding 1 mg of purified PvdQ (1 mg/ml) protein, and incubating 4 h at30°C. The mixture was again separated using the same SPE process de-scribed above for PVDI, but the 50% methanol fraction was collected, airdried, resuspended in 1 ml H2O, and stored at �20°C till further analysis.

(iv) PVDI(L-Glu). PVDI(L-Glu) was obtained by mixing the 1 ml ferrib-actin-containing solution with 50 ml of 50 mM CHES (N-cyclohexyl-2-aminoethanesulfonic acid) buffer and 250 �M CuSO4 at pH 9 and 1 mg ofpurified PvdP and incubating the mixture for 4 h at 30°C. The sample wasagain separated using the same SPE process described for ferribactin, airdried, resuspended in 1 ml of H2O, and stored at �20°C till further anal-ysis.

Analysis of PVDI precursor molecules. To verify the identity of thereaction products ferribactin and PVDI(L-Glu), the samples were analyzedby isoelectric focusing and liquid chromatography-mass spectrometry(LC-MS). The concentrations of the samples were calculated after mea-suring their dry weight and considering all the samples (with the excep-tion of PVDI) of a high purity as determined by high-performance liquidchromatography (HPLC) and gas chromatography (GC)-MS analyses.

Isoelectric focusing. PVDI and its precursors [PVDIq, ferribactin,and PVDI(L-Glu)] were separated based on their isoelectric point (pI) usingisoelectric focusing (IEF) gels (8, 20, 21) at pH 3.5 to 9.5 (Invitrogen).PVDI, PVDIq, ferribactin, and PVDI(L-Glu) were aliquoted in differenttubes containing the same concentration of the starting material (�1mg/ml) to ensure that the properties displayed in the IEF gels were notbiased by concentration differences. After separation, gels were visualizedunder UV light to detect the formation of the fluorescent chromophore.Here, the IEF gel was overlaid with a fresh 1-mm CAS agarose gel tillorange bands, indicative of iron chelation, developed (20).

Fluorescence spectrometry. A PVDIq sample, obtained as previouslydescribed, was divided into 3 aliquots and resuspended in 2 ml of either100% methanol (in the case of PVDIq due to its low solubility) or 0.1 Macetate buffer (pH 5). The final concentration of PVDIq was 1 mg/ml in allthree aliquots. A total of 20 �l of purified PvdQ (1 mg/ml) was added toone of the samples to obtain ferribactin. A total of 20 �l of PvdQ (1mg/ml) and 10 �l of purified PvdP (2 mg/ml) were added to the secondsample. No additional enzymes were added to the third PVDIq sample.Additionally, a fourth sample containing PVDI isolated from the super-natant of P. aeruginosa PAO1 was also tested as a control. CuSO4 wasadded to all 4 samples to a final concentration of 250 �M, after which thesamples were incubated for 24 h at room temperature prior to the analysis.PVDIq, ferribactin, PVDI(L-Glu), and PVDI were analyzed using a VarianCary Eclipse fluorescence spectrophotometer. Emission was scannedfrom 400 to 600 nm after excitation at a wavelength of 390 nm. Theexcitation and emission slit was set at 10 nm, and the scan was performedat a rate of 300 nm/min, with an average time of 0.1 s and a data interval of0.5 nm. FeCl3 was added to each of the samples at various final concen-trations (10 �M, 20 �M, 200 �M, and 400 �M) to determine the ability ofeach molecule to bind iron and consequently quench fluorescence (seeFig. S1 in the supplemental material).

LC-MS. Liquid chromatography-electrospray mass spectrometry wasperformed on an API3000 triple quadrupole mass spectrometer (PESciex), equipped with a turbo ion spray source coupled to a Shimadzu LCsystem equipped with LC-20AD gradient pumps and a SIL-20AC au-tosampler. Analyst 1.5.1 software was used for data acquisition and eval-uation. Each sample containing approximately 1 mg/ml (suspended in100% methanol) was diluted 20 times in methanol before injection, and30 �l was injected on a C18 Phenomenex Gemini 5-�m, 110-Å, 250- by4.6-mm column. The gradient mobile phase composition was a mixtureof solvent A (consisting of 99.9% H2O and 0.1% formic acid) and solventB (consisting of 50% acetonitrile and 50% acetone). The main flow was set

FIG 2 Structure of ferribactin (A) and its product, PVDI(L-Glu) (B), after in-cubation with PvdP.

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at 0.6 ml/min, and an additional postcolumn flow was added, providing aflow of 0.2 ml/min of solvent D (consisting of 99.9% acetonitrile and 0.1%formic acid) to improve spray conditions. Elution was performed using a25-min linear gradient from 5% to 90% solvent B.

Enzymatic assays. Functional prediction based on the fold algorithmrecognition server FFAS (22–24) suggested that PvdP shares homologywith polyphenol oxidases and tyrosinases. The activity of PvdP was mon-itored at 30°C over a range of substrates (0.5 to 6.5 mM) that includedL-Tyr, D-Tyr, and L-Dopa (Sigma-Aldrich) by measuring A475 for the for-mation of dopachrome (ε 3,600 M�1 cm�1) and ferribactin by mea-suring A405 (25) for the formation of the PVDI chromophore (ε 4,160M�1 cm�1). A range of pH (3 to 12) was tested using 50 mM CHES and 10mM phosphate buffers to determine optimal conditions for enzyme ac-tivity. All measurements were performed at 30°C. The optimal pH of thereaction mixture was determined to be 9, and all subsequent kinetic ex-periments were performed in 50 mM CHES buffer at pH 9. All reactionswere performed in the presence of 250 �M CuSO4, which was determinedto be the optimal concentration for enzyme activity. Mushroom tyrosi-nase (T3824-25KU; Sigma-Aldrich) was used as a control to evaluate thespecificity of PvdP toward ferribactin.

Kinetic studies were performed at 30°C in 50 mM CHES buffer and250 �M CuSO4 at pH 9. Under these conditions, substrate concentrationsranging from 0.5 to 6.5 mM were incubated with 22.5 �g of PvdP in a 1-mlvolume, and the absorbance was monitored every 2 min at either 475 nmfor L-Tyr, D-Tyr, and L-Dopa or 405 nm for ferribactin. In the case ofL-Tyr, D-Tyr, and L-Dopa, kinetic parameters were obtained by applyingthe Michaelis-Menten equation (v Vmax � S/Km � S), with v rate ofreaction, Vmax maximum velocity, Km Michaelis-Menten constant,and S substrate concentration. In the case of ferribactin, kinetic param-eters were calculated using the substrate inhibition equation (26, 27),where v Vmax/[1 � (Km/S) � (S/Ksi)], with Ksi representing the con-stant describing the substrate inhibition interaction. The reaction rateswere corrected for the rate of the spontaneous reaction.

PvdP structural model. A PvdP model was created using the sameFFAS03 server that was used to predict its protein function through a foldrecognition algorithm. PvdP was modeled based on the closest homo-logue determined by the server, which corresponded to the arthropodanhemocyanin from the California spiny lobster Panulirus interruptus (Pro-tein Data Bank [PDB] file 1HC1). Given the low amino acid identitybetween these two proteins, minimization on the PvdP model was per-formed using Discovery Studio 3.0 (Accelrys, USA), consisting of 1,000steps of steepest descent followed by 5,000 iterations of the adopted basisset Newton-Raphson algorithm using an energy tolerance of 0.001 kcal ·mol�1 · Å�1 to reduce as much as possible the errors.

Site-directed mutagenesis and analysis of PvdP mutants. PvdPstructure model suggested His216, His220, His271, His375, His379, andHis432 as the six histidine residues forming the di-copper active site.Protein sequence alignment of PvdP and protein homologues in otherfluorescent pseudomonads was performed using an identity score matrix(Blosum62) in Geneious 7.0.6 (see Fig. S2 in the supplemental material).Using standard methods (28), site-directed mutagenesis was performedto substitute each individual histidine to an aspartate. Enzymatic activityof each mutant enzyme was assayed as described in the previous section.Circular dichroism (CD) was performed using a Jasco J-715 CD spectro-photometer equipped with a PFD350S Peltier temperature-control unit(Jasco). Rectangular quartz 1-mm path length cuvettes were used. Proteinsamples were dialyzed against PBS (pH 7.3) and adjusted to a final con-centration of 1 mg/ml. Wavelength spectra were recorded between 250and 205 nm using a 0.2-nm step size and 1-nm bandwidth at 25°C for thewild type and each of the mutant proteins to verify secondary structureintegrity.

RESULTSPvdP is a copper-dependent tyrosinase. During a bioinformaticssearch, using the “fold and function assignment system” (FFAS)

algorithm (22–24) to determine the function of the uncharacter-ized proteins predicted to be involved in the maturation of thePVDI chromophore of P. aeruginosa (PvdM, PvdN, PvdO, andPvdP), we obtained several hits for PvdP that were all linked toclosely related proteins: hemocyanin, phenoloxidase, and tyrosi-nase. Despite the low amino acid identity between PvdP and theseproteins (7 to 12%), the presence of a D-tyrosine in the immaturechromophore of the PVDI precursor ferribactin (29) prompted usto investigate whether PvdP might have tyrosinase activity. Afterproduction in Escherichia coli, purified PvdP was tested in a stan-dard spectrophotometric assay for tyrosinase activity. In a firstassay with L- and D-tyrosine, the presence of PvdP induced a weakbrown coloration at A475 typical for the formation of melanin.Since tyrosinases are characterized by a type 3 copper center (30,31), and given the slow reaction rate in our preliminary assay, wedecided to titrate different amounts of copper. Copper titrationrevealed a 10-fold increase in activity on L- and D-tyrosine in thepresence of 250 �M CuSO4 while decreasing at higher concentra-tions (see Fig. S3 in the supplemental material), demonstratingthat PvdP requires copper as a cofactor.

PvdP is a tyrosinase essential for chromophore maturationin PVDI synthesis. In our search for the substrate of PvdP in thePVDI synthesis pathway, we tested a pvdP-knockout strain, the P.aeruginosa PAO1 pvdP::Kn strain (32), and tried to isolate thesubstrate for PvdP. Unfortunately, we were unable to isolate andpurify this compound from this strain due to low quantities andmultiple forms of PVDI precursors. In an attempt to overcomethis problem, we used another P. aeruginosa mutant, the P. aerugi-nosa PAO1 �pvdQ mutant, as starting material to obtain the C14-acylated precursor, PVDIq (see Materials and Methods). Subse-quently, PVDIq was converted in vitro with purified PvdQ andisolated. This method allowed us to produce semipreparativeamounts of product, with both high yield and purity. In accor-dance with earlier publications (7, 10, 11), the product was veri-fied to be ferribactin using LC-MS analysis (Fig. 3) and other tests(see below).

The biosynthetic route from ferribactin to the chromophore ofPVDI has been suggested to involve several oxidation steps (7, 33)but so far no enzyme responsible has been identified. Incubationof ferribactin with PvdP resulted in an increase of A405, indicativeof the formation of the mature PVDI chromophore. Incubation ofcommercially available mushroom tyrosinase together with ferri-bactin did not result in any changes of A405 even after 24 h ofincubation. The maturation reaction as proposed by Dorrestein etal. (33) involves 3 oxidation steps and shows high similarity to thetyrosinase activity on L-Tyr and L-Dopa to dopachrome (Fig. 4).The incubation of ferribactin with purified PvdP resulted in anabsorbance increase at 405 nm, appearance of fluorescence, andan increase in iron chelation capability (Fig. 5). We named thisproduct PVDI(L-Glu) to differentiate it from PVDI isolated from P.aeruginosa PAO1 and PA14 that contain different substitutions ofthe L-Glu chain presumably as a consequence of the enzymaticreaction with other PVD enzymes of as-yet-unknown function.Importantly, whereas ferribactin is a nonfluorescent substrate, theproduct of the PvdP-catalyzed reaction is a green fluorescent mol-ecule; thus, the reaction proceeds past the nonfluorescent dihy-droPVD (34). All products have been analyzed by different assaysfor identification (see below).

PvdP is a periplasmic protein requiring a TAT secretion sys-tem. PvdP has been proposed to be a periplasmic protein due to

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FIG 3 Mass spectra of ferribactin (eluting between 3.204 and 3.04 min on a C18 Phenomenex Gemini column [5 �m, 110 Å, 250 by 4.6 mm]) (a) and PVDI(L-Glu)

(eluting between 2.937 and 3.137 min) (b). The calculated uncharged masses of ferribactin and PVDI(L-Glu) are 1,351.2 Da and 1,364.5 Da, respectively. Noticethat PVDI(L-Glu) carries already one positive charge in the chromophore of PVDI(L-Glu) (Fig. 2).

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the presence of a putative TAT signal peptide (35, 36). To verifythis, we constructed the plasmid pME-pvdP_H6 and transformedit into the E. coli HB2151 WT and the E. coli HB2151 �tatC mutantand analyzed the periplasmic and cytoplasmic fractions of eachstrain for the presence of PvdP_H6. The absence of PvdP in theperiplasmic fraction of the E. coli HB2151 �tatC mutant (Fig. 6)clearly demonstrates that PvdP is a periplasmic protein that re-quires a TAT secretion system for its secretion to the periplasm. Asmall fraction of PvdP remains in the cytoplasm of the E. coliHB2151 WT, which may be a consequence of the overexpressionof this protein and consequent saturation of the TAT system.

Analysis of products PVDI, PVDIq, ferribactin, andPVDI(L-Glu). The spectral properties of the four products PVDIq, fer-ribactin, PVDI(L-Glu), and PVDI were examined by fluorescence spec-troscopy. Although PVDIq and ferribactin show some fluorescenceat 450 nm, the fluorescence of PVDI(L-Glu) and PVDI is 35-foldhigher than that of PVDIq and ferribactin (see Fig. S1 in the sup-plemental material). The latter two fluorescent products have anearly identical spectrum, in line with the expectation that theyhave a very similar structure. The addition of ferrous iron resulted

in fluorescence quenching, indicating that all products can bindFe3�. Due to the very high affinity of PVD for ferrous iron, thisassay does not allow to efficiently compare the affinities of thedifferent species.

In parallel, we have examined these same molecules using sid-erotyping. This technique allows us to visualize siderophores andclassify bacterial strains by the study of the migration behavior oftheir respective siderophores (8, 20, 21). This technique was ex-ploited to analyze the different properties of PVDI as isolated fromP. aeruginosa PAO1, in comparison to the three compounds iso-lated in our experiments: PVDIq, ferribactin, and PVDI(L-Glu). UVillumination demonstrates that PVDIq and ferribactin lack thefluorescence typical of PVDIs (Fig. 5a), in line with the absence ofa mature chromophore. Clearly, PVDI and PVDI(L-Glu) do showfluorescence, and one of the bands runs at comparable height inthe gel, suggesting these compounds to be very similar. In the CASoverlay, PVDI and PVDI(L-Glu) show a band at the same height inthe IEF gel, with very similar staining (Fig. 5b), demonstrating acomparable, strong Fe3� binding capacity. These results stronglysuggest that these two compounds are very similar if not identical.

FIG 4 Comparison of the tyrosinase reaction leading from tyrosine to dopachrome (A) versus the same reaction leading from ferribactin to PVDI (B). Theanalogy between the two reactions provides an explanation to the formation of the third ring in the PVDI chromophore.

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The weaker staining with ferribactin suggests a lower affinity forFe3�, in line with the assumption that this compound equals thelow affinity molecule ferribactin (34). Obviously, iron staining ishardly present in the compound PVDIq (Fig. 5b). We hypothesizethat this feature is a consequence of the PVDIq structural confor-mation where the immature chromophore may be adapting a dis-tant position from the other two iron binding groups as a result ofthe attached C14-acyl chain (11).

The compounds ferribactin and PVDI(L-Glu) were also analyzedby LC-MS. The mass for ferribactin was calculated based on the

previously published structures (7, 29) and considering the acylchain (often represented as –R due to the different possible sub-stitutions) to be the original glutamate present after NRPS synthe-sis. Similarly, the mass of PVDI(L-Glu) was calculated from ferrib-actin using the reaction scheme in Fig. 4B. The results of theLC-MS analysis (Fig. 3) demonstrate that the experimentally de-termined molecular mass of ferribactin, 1,351.2 Da, correspondsto the calculated mass of 1,351.42 Da. Similarly, the calculated andexperimental molecular masses of PVDI(L-Glu) are practically iden-tical: 1,364.4 and 1,364.5 Da. These results strongly underline ourinterpretation of the catalytic activity of PvdQ and PvdP in thePVDIq and ferribactin maturation processes, respectively.

Kinetic analysis of PvdP. The kinetic parameters of the PvdP-catalyzed reactions were measured for L-Tyr, D-Tyr, L-Dopa, andferribactin. The enzyme exhibited typical Michaelis-Menten ki-netics on L-Tyr, D-Tyr, and L-Dopa and substrate inhibition onferribactin with a Ksi of 3.37 � 0.54. The Km and kcat values do notdiffer much for the various substrates, with the exception of asignificantly lower kcat value for L-Dopa (Table 1).

PvdP structural model. Modeling the active site of PvdP basedon its closest structure-solved homologue (PDB file 1HC1) revealsthe presence of the six histidines (His216, His220, His271, His375,His379, and His432) presumably responsible for the formation ofthe characteristic di-copper active site of tyrosinases (Fig. 7). Toverify this, we constructed single amino acid mutants where eachof the six histidines was substituted with an aspartic acid andtested the resulting proteins for enzymatic activity toward tyrosine

FIG 5 UV and CAS overlay detection of siderophores after migrating throughan IEF gel. (a) Fluorescence under UV light of PVDI, PVDIq, PVDIq incubatedwith PvdQ (ferribactin), and PVDIq digested with PvdQ-PvdP [PVDI(L-Glu)].(b) After detection of the fluorescent chromophores, the gel was covered with1 mm of CAS agarose, revealing orange spots that indicate iron-chelating ac-tivities of PVDI and its precursors.

FIG 6 Periplasmic localization and TAT secretion system dependency ofPvdP. After separating the cytoplasmic and periplasmic proteins, PvdP_H6was detected using an anti-6�His antibody. After cell fractionation, PvdP_H6was detected in an E. coli wild-type strain having a functional TAT system andin a �tatC mutant. GroEL detection was used as a control for the cytoplasmicfraction. PvdP_H6 is absent in the periplasmic fraction of the E. coli HB2151�tatC mutant (1st lane, right side), indicating the requirement of the TATsystem for the periplasmic localization of PvdP. A large amount of PvdP can bedetected in a wild-type E. coli, confirming that PvdP is a periplasmic protein(1st lane, right side). Coomassie staining served as the loading control.

TABLE 1 Kinetic values of PvdP

Substrate Km (mM) kcat (s�1)kcat/Km

(mM�1 s�1)

L-Tyr 1.3 � 0.05 1.38 � 0.09 1.06D-Tyr 2.2 � 1.65 0.77 � 0.33 0.35L-Dopa 0.75 � 0.28 0.13 � 0.01 0.17Ferribactin 1.6 � 0.23 1.30 � 0.0 0.81

FIG 7 Model of the active site of PvdP obtained using the FFAS03 server afterminimization with Discovery Studio 3.0. The 6 histidines involved in copperbinding are highlighted in yellow.

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or ferribactin. Mutated proteins behaved identically to WT PvdPduring purification. In addition, the presence of secondary struc-tures in each purified mutant protein was checked by circulardichroism and showed results comparable to those of WT PvdP,indicating that the mutations had no effect on the structural in-tegrity of the proteins. None of the mutants demonstrated tyrosi-nase activity against any of the previously tested substrates. Theseresults are in agreement with the proposed structural model thatindicates that these six histidines are essential to form the di-cop-per active site of PvdP.

PvdP is the first member of a tyrosinase subfamily devoted toPVDI maturation. The small percent identity between PvdP andother tyrosinases and the exceptionally large size of its substratesuggest that PvdP is the first characterized tyrosinase member of aprotein family exclusive to fluorescent pseudomonads. In P.aeruginosa and probably other pseudomonads species, PvdP isdedicated to the maturation of PVD and PVD-like siderophores.To confirm this hypothesis, we performed a phylogenetic analysisusing the amino acid sequence of the closest PvdP homologues (asdetermined by a protein BLAST) and representing one singlePvdP sequence of each representative fluorescent pseudomonadand a few examples outside this clade. Our results (Fig. 8) confirmthat PvdP (as other PVDI enzymes) is absent in nonfluorescentpseudomonads, with the closest homologue outside the fluores-cent pseudomonads group sharing less than 40% identity. Thelevel of identity between PvdP and homologues in other fluores-cent pseudomonads is high (51 to 66%) (Fig. 8) but still divergent,which may be related to the structural differences in the PVDsproduced in different species (8, 32, 37) that during evolutionwould have driven PvdP toward a coevolutive process in order tobetter fit each molecule. Furthermore, the absence of activity ofcommercial mushroom tyrosinase on ferribactin (data notshown) underlines the hypothesis that this new family of PvdP-like tyrosinases has evolved to accommodate large peptidic sub-strates and accomplish its function on PVDI biosynthesis. Analignment of the protein sequences used for the phylogenetic anal-ysis reveals that all six histidines involved in the copper binding arestrictly conserved in all fluorescent pseudomonads (see Fig. S2 inthe supplemental material).

DISCUSSION

Previous studies have shown the involvement of PvdP in pyover-dine biosynthesis (32); however, the exact function of PvdP hasremained elusive. Its low homology to other annotated proteins isevident when using a basic BLAST sequence analysis. In the pres-ent study, by using the FFAS tool (22–24) that predicts proteinfunction based on folding, we were able to obtain several hits,suggesting that PvdP could act as a tyrosinase. We pursued thishypothesis encouraged by the presence of a D-tyrosine in the PVDIprecursor, ferribactin (29), in the exact location where the PVDIchromophore is located. Looking at the previously describedproducts being formed from tyrosine in the consecutive steps of atyrosinase reaction, we further hypothesized that the same reac-tion on ferribactin would lead to the formation of the PVDI chro-mophore (Fig. 4A and B). Purified PvdP was then tested againstboth L-Tyr and D-Tyr. As expected, PvdP transformed these twocolorless substrates into a dark-brown compound indicative of amelanization reaction typical of tyrosinases, in which after theenzymatic conversion of tyrosine to L-Dopa and subsequentlydopaquinone, a series of spontaneous reactions takes place, lead-ing to the formation of melanin. Despite the positive result, thelow activity of PvdP on these substrates was surprising. We hy-pothesized that the two copper atoms present in all tyrosinasesand responsible for the activity of these proteins (30) may not bepresent in all of our purified PvdP. This could either be a conse-quence of the large amounts of PvdP generated after overexpres-sion in E. coli or of the absence of copper in the PvdP fraction thatis still in the cytoplasm, presumably due to a saturation of the TATpathway after PvdP overexpression. The activity of our batch ofpurified PvdP was then tested in the presence of various amountsof CuSO4, revealing a maximum activity toward tyrosine around125 to 250 �M (see Fig. S3 in the supplemental material). It seemsthat the copper concentrations in these assay conditions are ide-ally balanced. Concentrations higher than 250 �M seem to disturbthe interaction between the substrate and the metal, either bycompetition for the oxygen site, a phenomenon that is often seenfor metalloproteins, or by competitive inhibition where the cop-per-bound substrate would be unable to fit in the copper-boundactive site of PvdP. These results confirmed our hypothesis andalso provide an explanation for the previously reported require-ment for copper for maximum PVDI production in P. aeruginosa(38).

The next step was to analyze the reaction of PvdP on the PVDprecursor in P. aeruginosa from the cellular and extracellular frac-tions of a P. aeruginosa pvdP::Kn mutant (32). Our attempts tofind this molecule proved unsuccessful due to the low concentra-tion and the diversity of other similar molecules obtained in this P.aeruginosa mutant. To overcome this problem, we decided to pu-rify a previous substrate (PVDIq), which had been extensivelystudied in our lab and reported by others (11, 12). We obtainedferribactin by digesting PVDIq with PvdQ protein, which wasused as a template to test the activity of PvdP. A reaction of ferri-bactin with PvdP transformed the initially faint yellow moleculeinto a fluorescent green molecule with a strong absorption at A405

nm, confirming our prediction that PvdP was responsible for theformation of the PVDI chromophore. While this approach al-lowed us to corroborate the function of PvdP in the PVDI path-way, we are unable to propose ferribactin as the natural substrateof PvdP, since other proteins, such as PvdM, PvdN, and PvdO,

FIG 8 Phylogenetic tree of PvdP homologues from fluorescent pseudomon-ads (Azotobacter vinelandii, Cellvibrio japonicus, Pseudomonas chlororaphis,Pseudomonas aeruginosa, Pseudomonas entomophila, Pseudomonas fluore-scens, Pseudomonas putida, Pseudomonas synxantha, Pseudomonas savas-tanoi, and Pseudomonas syringae) and the three closest protein homologuesoutside the Pseudomonadales clade (Rhodopirellula baltica, Bacillus atrophaeus,and Geodermatophilus obscurus). Numbers in red indicate percentages ofamino acid identity related to P. aeruginosa PAO1 PvdP.

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with an as-yet-unassigned function, may be converting ferribactininto yet another product prior to PvdP. For this reason, we namedPVDI(L-Glu) the product of the reaction of ferribactin plus PvdP.

The identity of the molecules was verified using LC-MS: thepeaks corresponding to ferribactin and PVDI(L-Glu) were foundwith masses almost identical to those calculated: 1,351.2 Da com-pared to the calculated a mass of 1,351.42 Da in the case of ferri-bactin and 1,364.4 compared to the calculated mass of 1,364.5 Dafor PVDI(L-Glu) (Fig. 3a and b).

Fluorescence spectrometry was performed on all four com-pounds [PVDIq, ferribactin, PVDI(L-Glu), and PVDI] to evaluatethe fluorescence properties of each molecule. We compared theirfluorescence after PvdP addition and tested their ability to bindiron by testing fluorescence quenching. The results obtainedclearly indicate that despite all sharing a similar fluorescent pro-file, there is a 35-fold increase in fluorescence in PVDI(L-Glu) andPVDI compared to the other two PVD precursors. Unfortunately,this technique did not allow us to establish whether PVDI(L-Glu)

and PVDI have a higher affinity for iron as a consequence of theformation of the PVD chromophore due to the high differences intotal fluorescence among the molecules. To overcome this, wedecided to use siderotyping (8, 20, 39). This technique allows us tocompare the four molecules according to their different migrationon an IEF gel and examine their fluorescence and iron bindingproperties (Fig. 5). Both PVDIq and ferribactin revealed undetect-able fluorescence under a standard UV light in accordance withthe absence of a chromophore in these molecules. AdditionallyPVDI(L-Glu) displayed a stronger coloration on the same gel thanferribactin, a clear indication of the increase in affinity for Fe3� ofthis molecule as a consequence of the formation of the third ironbinding group (Fig. 2A and B). Kinetic analysis of PvdP activitytoward the different substrates revealed that PvdP is neither spe-cific for our proposed natural substrate (ferribactin) nor its targetamino acid D-Tyr. Nevertheless, time of expression (13) and cel-lular localization (periplasm) may be the reason a higher activityof PvdP toward ferribactin is not required. Tyrosine is synthesizedin the cytoplasm, where it is used in protein synthesis, while PvdPas a Cu2�-dependent enzyme will be active only once it hasreached the periplasm, where it can convert ferribactin. In com-parison to PvdP, commercially available mushroom tyrosinasedid not show activity on ferribactin even after 24 h of incubation.This strongly supports the hypothesis that PvdP belongs to a newfamily of tyrosinases. Phylogenetic analysis suggests that PvdP is amember of a group of tyrosinases, each of which may be involvedin the maturation of their canonical PVDI chromophore.

PVD siderophores require a large array of proteins for theirsynthesis, transport, and maturation. A full elucidation of thecomplete PVDI pathway is crucial to understand the chemicalnature of this complex siderophore. Additionally, since iron is oneof the most important limiting factors for bacterial growth, inter-fering with siderophore production may contribute to the devel-opment of novel antimicrobial agents capable of interfering withsiderophore synthesis. The addition of PvdP to the list of enzymeswith an assigned reaction in the biosynthesis of PVDI leaves theconversion of the attached L-Glu side chain into the various sidechains observed in PVDI (40) as the only reactions left for theremaining uncharacterized enzymes, e.g., PvdM, PvdN, andPvdO.

ACKNOWLEDGMENTS

This work was partly funded by EU grant Antibiotarget MEST-CT-2005-020278 to P.N.-J., G.K., and W.J.Q.

We are grateful to Isabelle J. Schalk and Herbert Budzikiewicz foruseful discussion and analysis of the PVDI maturation and synthesis me-tabolites, Ian Lamont for providing us with the P. aeruginosa PAO1PvdP::Kn strain, Wesley R. Browne for assistance with the circular dichr-oism analysis, Lígia O. Martins and Vânia Brissos for assistance with thefluorescence measurements, and Jessica Thompson and Hjalmar P. Per-mentier for carefully reading the manuscript.

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