Unsaturated Macrocyclic Dihydroxamic Acid Siderophores Produced

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Unsaturated Macrocyclic Dihydroxamic Acid Siderophores Produced by Shewanella putrefaciens Using Precursor-Directed Biosynthesis Cho Z. Soe and Rachel Codd* School of Medical Sciences (Pharmacology) and Bosch Institute, The University of Sydney, Sydney, New South Wales 2006, Australia

ABSTRACT: To acquire iron essential for growth, the bacterium Shewanella putrefaciens produces the macrocyclic dihydroxamic acid putrebactin (pbH2; [M + H+]+, m/zcalc 373.2) as its native siderophore. The assembly of pbH2 requires endogenous 1,4diaminobutane (DB), which is produced from the ornithine decarboxylase (ODC)-catalyzed decarboxylation of L-ornithine. In this work, levels of endogenous DB were attenuated in S. putrefaciens cultures by augmenting the medium with the ODC inhibitor 1,4-diamino-2-butanone (DBO). The presence in the medium of DBO together with alternative exogenous non-native diamine substrates, 15N2-1,4-diaminobutane (15N2-DB) or 1,4-diamino-2(E)-butene (E-DBE), resulted in the respective biosynthesis of 15N-labeled pbH2 (15N4-pbH2; [M + H+]+, m/zcalc 377.2, m/zobs 377.2) or the unsaturated pbH2 variant, named here: E,E-putrebactene (E,E-pbeH2; [M + H+]+, m/zcalc 369.2, m/zobs 369.2). In the latter system, remaining endogenous DB resulted in the parallel biosynthesis of the monounsaturated DB-E-DBE hybrid, E-putrebactene (E-pbxH2; [M + H+]+, m/zcalc 371.2, m/zobs 371.2). These are the first identified unsaturated macrocyclic dihydroxamic acid siderophores. LC−MS measurements showed 1:1 complexes formed between Fe(III) and pbH2 ([Fe(pb)]+; [M]+, m/zcalc 426.1, m/zobs 426.2), 15N4pbH2 ([Fe(15N4-pb)]+; [M]+, m/zcalc 430.1, m/zobs 430.1), E,E-pbeH2 ([Fe(E,E-pbe)]+; [M]+, m/zcalc 422.1, m/zobs 422.0), or EpbxH2 ([Fe(E-pbx)]+; [M]+, m/zcalc 424.1, m/zobs 424.2). The order of the gain in siderophore-mediated Fe(III) solubility, as defined by the difference in retention time between the free ligand and the Fe(III)-loaded complex, was pbH2 (ΔtR = 8.77 min) > E-pbxH2 (ΔtR = 6.95 min) > E,E-pbeH2 (ΔtR = 6.16 min), which suggests one possible reason why nature has selected for saturated rather than unsaturated siderophores as Fe(III) solubilization agents. The potential to conduct multiple types of ex situ chemical conversions across the double bond(s) of the unsaturated macrocycles provides a new route to increased molecular diversity in this class of siderophore.

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B (log K 30.8)12,13 has found clinical use to treat chronic iron overload disease, which occurs as a secondary complication of transfusion-dependent blood disorders.14,15 The affinity of siderophores toward Fe(III) and other transition metal ions makes these valuable ligands for medicine and environmental metal ion remediation.16−23 About one or two new bacterial siderophores or variants of previously identified siderophores are isolated per year.24−30 To add to the structural diversity in nature, new siderophore analogues can be accessed using precursor-directed biosynthesis, in which culture medium is augmented with non-native substrates for uptake by the regular biosynthetic pathway of the bacterium.31−34 This approach can be high risk, since it assumes that the components added to the

n an aqueous, aerobic, and pH neutral environment, the bioavailability of essential Fe is restricted, since it exists predominantly as insoluble Fe(III)-oxy-hydroxide complexes. In order to acquire sufficient Fe for growth, bacteria have evolved to produce a class of high-Fe(III)-affinity, lowmolecular-weight ( E-pbxH2 (ΔtR =

6.79 min) > E,E-pbeH2 (ΔtR = 5.53 min), was the same as observed in the case of Fe(III). Molecular Modeling. Molecular modeling was undertaken to explore the relative energies of the saturated and unsaturated 950

dx.doi.org/10.1021/cb400901j | ACS Chem. Biol. 2014, 9, 945−956

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Figure 4. Selected-ion monitoring (SIM) of signals attributable to protonated adducts of the free ligands E,E-pbeH2, E-pbxH2 or pbH2 (a) (m/z 369, 371 or 373, respectively) and the corresponding 1:1 Fe(III) complexes, as (b) molecular ions (m/z 422, 424, 426, respectively) or (c) solvated (0.5H2O·CH3OH) molecular ions (m/z 463, 465, 467, respectively). The signal intensities within each ion type have been normalized to 100%. The sum of the signals in (b) and (c), which represents the total concentration of the relevant Fe(III) complex, is shown in (d).

Figure 5. Experimental (black) and simulated (white) MS isotope patterns of (a) [Fe(pb)]+, (b) [Fe(15N4-pb)]+, or (c) a mixture of [Fe(E,E-pbe)]+ and [Fe(E-pbx)]+ (0.05:0.95 ratio), with the latter as sampled from the LC peak from Figure 3 at tR 23.63 min. The five panels below (c) represent data sampled from the same LC peak at tR 23.95, 24.11, 24.21, 24.37, or 24.47 min, with the [Fe(E,E-pbe)]+: [Fe(E-pbx)]+ ratio determined from simulation. The data has been normalized to the signal attributable to the major [M]+ ion.

complexes and to examine the energies of theoretical analogues assembled from 1,4-diamino-2(Z)-butene (Z-DBE). The structure of [Fe(pb)(OH2)2]+ was built from the X-ray crystal structure of [Fe2(alcaligin)3],68 with aqua ligands positioned at the coordinates of the oxygen donor atoms of the bridging alcaligin ligand. Of the 1:1 Fe(III)-ligand complexes, [Fe(pb)(OH2)2]+ had the lowest energy (513.8 kJ mol−1) (Table 3). The presence of one or two E-configured double bonds resulted in increased energies in [Fe(E-pbx)(OH2)2]+ (532.6 kJ mol−1) and [Fe(E,E-pbe)(OH2)2]+ (553.1 kJ mol−1), with higher energies for the equivalent Z-isomers [Fe(Z-pbx)(OH 2 ) 2] + (552.7 kJ mol −1 ) and [Fe(Z,Z-pbe)(OH 2 ) 2 ] + (560.6 kJ mol−1). The higher energy values of the Z-DBEbased isomers are attributable to higher steric strain inherent to the unsaturated ring system and a number of close intra-atomic contacts that manifest from the restricted conformational flexibility about the amide region (Table 3, Figure 8). At neutral pH values, 2:3 Fe(III):pbH2/alcaligin complexes are formed, which feature two Fe(III) centers that are coordinated by a discrete tetradentate ligand and a third ligand that bridges the metal ions.40,47,68 A model of the structure of [Fe2(pb)3], based on [Fe2(alcaligin)3],68 showed a high similarity with the isomer [Fe2(E,E-pbe)2(E,Z-pbe)], in which each E,E-pbeH2 unit coordinates a discrete Fe(III) ion and E,ZpbeH2 acts as the bridging ligand (Figure 9). The RMS error for the overlay of [Fe2(pb)3] and [Fe2(E,E-pbe)2(E,Z-pbe)] (excluding H atoms) was 0.442 Å. This indicates that the preferred 2:3 complex between Fe(III) and a diunsaturated

macrocyclic dihydroxamic acid would form from 2 molar equiv of E,E-pbeH2 and 1 molar equiv of E,Z-pbeH2. Conclusion. This work has shown that S. putrefaciens biosynthesizes mono- and diunsaturated macrocyclic dihydroxamic acids upon augmenting culture medium with DBO and 1,4-diamino-2(E)-butene (E-DBE). This strategy yielded E,E-putrebactene (E,E-pbeH2) and E-putrebactene (E-pbx), the latter of which was formed from one unit of E-DBE and one unit of endogenous 1,4-diaminobutane (DB). This is the first example of the use of a Shewanella species for generating new compounds via precursor-directed biosynthesis. The integrity of diamine uptake and siderophore biosynthesis in S. putrefaciens was evident in the production of 15N4-labeled putrebactin (15N4-pbH2) from medium augmented with DBO and 15N2-DB. Culture supernatant was subject to multiple chromatographic steps, and the siderophores present in the final purified extracts were characterized by LC−MS as native and Fe(III)- or Ga(III)-loaded solutions. Each of E,E-pbeH2 and E-pbxH2 remained functional as Fe(III) ligands, with the formation of 1:1 complexes [Fe(E,E-pbe)]+ and [Fe(E-pbx)]+ at low pH values. The increase in the siderophore-mediated 951



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Figure 6. Reverse-phase HPLC trace (absorbance 220 nm, panel at left) and positive ion mass spectrometry at retention times for the major species (panels at right), from a semipurified extract of S. putrefaciens supernatant that was cultured in the (a) absence of exogenous diamine (native system) or the presence of (b) 10 mM DBO and 10 mM 15N2-DB or (c) 10 mM DBO and 10 mM E-DBE. The extract was analyzed in the presence of exogenous Ga(III). The gradient in (a) was the same in (b) and (c) and has been omitted for clarity.

solubility of Fe(III) correlated with the degree of saturation in the siderophore: pbH2 > E-pbxH2 > E,E-pbeH2, which may explain in part why nature has evolved saturated siderophores, with this interpretation based on data obtained under acid conditions. These results show that the PubABC enzymes involved in pbH2 biosynthesis in S. putrefaciens have substrate specificities beyond DB, with PubA competent in the mono-N-hydroxylation of E-DBE and PubB or PubC competent in the transformation of N-hydroxy-E-DBE or N-hydroxy-N-succinylE-DBE, respectively (Scheme 3). Augmenting S. putrefaciens culture medium with DBO and 1,4-diamino-2(Z)-butene (Z-DBE) could furnish the Z-based analogues Z,Z-pbeH2 and Z-pbxH2, with the mixed system E,ZpbeH2 potentially accessible from augmentation with a mixture of E-DBE and Z-DBE. This assumes that the S. putrefaciens PubABC biosynthetic cluster could accept Z-DBE and downstream products as substrates. These experiments are ongoing in our group. The small differences between minimum energy values of Fe(III) complexes formed with E-DBE- or ZDBE-derived macrocycles suggests that each of Z,Z-pbeH2, ZpbxH2 and E,Z-pbeH2 would be competent Fe(III) ligands. These new unsaturated macrocyclic siderophores have potential in ex situ chemical oxidation reactions with OsO4 or KMnO4; or chloroperbenzoic acid, to produce syn- or antihydroxylated products, respectively.69,70 Through the insequence routes of precursor-directed biosynthesis and semisynthesis, this work describes new avenues toward the generation of a high degree of molecular diversity within the macrocyclic dihydroxamic acid class of siderophores.

Figure 7. Experimental (black) and simulated (white) MS isotope patterns of (a) [Ga(pb)]+, (b) [Ga(15N4-pb)]+, or (c) a mixture of [Ga(E,E-pbe)]+ and [Ga(E-pbx)]+ (0.08:0.92 ratio) with the latter as sampled from the LC peak at tR 23.79 min. The five panels below (c) represent data sampled from the same LC peak at tR 23.98, 24.15, 24.48, 24.55, or 24.74 min, with the [Ga(E,E-pbe)]+:[Ga(E-pbx)]+ ratio determined from simulation. The data has been normalized to the signal attributable to the major [M]+ ion.

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METHODS

Reagents and Chromatographic Resins. 1,4-Diamino-2-butanone dihydrochloride (DBO·2HCl, 99%), 15N2-1,4-diaminobutane dihydrochloride (15N2-DB·2HCl, 98% purity), FeCl3·6H2O (97%), and Ga(NO3)3·H2O (99.9%) were purchased from Sigma-Aldrich (St. Louis, MO). 1,4-Diamino-2(E)-butene dihydrochloride (E-DBE·2HCl, >96%) was purchased from Small Molecules, Inc. (Hoboken, NJ). Chromatography was carried out using XAD-2 resin (Amberlite), Sephadex LH-20 resin (Amersham Biosciences), Vivaspin C18 reverse-phase prepacked columns (12 cc) (Sartorius Stedim), and Ni(II) SepharoseTM 6 Fast Flow resin (GE Healthcare). Liquid Chromatography−Mass Spectrometry: Instrumentation. Liquid chromatography−mass spectrometry (LC−MS) was conducted on an Agilent series 1200 LC system with an Agilent 1260 Infinity binary pump and integrated vacuum degasser, autosampler, thermostatted column compartment and diode array detector, and an Agilent 6120 quadrupole mass spectrometer. The samples were analyzed on an Agilent C18 column (particle size 5 μm; 150 × 2.1 mm i.d.) with a gradient of 0−30% B over 40 min (A, H2O/formic acid 99.9:0.1; B, CH3CN/formic acid 99.9:0.1) and at a flow rate of 0.2 mL min−1. Agilent OpenLAB Chromatography Data System (CDS) ChemStation Edition was used for data acquisition and processing. 952



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Table 3. Energy Minima of pbH2 and New Experimental and Theoretical Macrocyclic Dihydroxamic Acid Siderophores distance (Å)b species

a

[Fe(pb)]+ [Fe(E,E-pbe)]+ [Fe(E-pbx)]+ [Fe(Z,Z-pbe)]+ [Fe(Z-pbx)]+ [Fe(E,Z-pbe)]+

−1

E (kJ mol )

Ox·O

Hx·O

Hx·O

Oy·OC

Hy·OC

Hy·ON

513.8 553.1 532.6 560.6 552.7 555.6

3.95 3.88 3.84 3.93 3.56 3.89

3.58 3.39 3.56 2.35 3.19 3.37

4.16 4.10 4.27 2.84 3.68 4.09

3.81 3.49 3.75 3.29 3.70 3.23

3.70 3.91 3.72 3.55 3.70 3.73

4.45 4.71 4.47 4.21 4.45 4.42

C

C

N

Calculations were undertaken on the diaqua species. bOx·OC denotes the distance between the O atom (amide) and the O atom of the proximal O(C) (hydroxamate) group. Hx·OC or Hx·ON denotes the distance between the H atom (amide) and the O atom of the proximal O(C) or proximal O(N) (hydroxamate) group, respectively. Subscripts x, y refer to the monomeric units of the (quasi)symmetric dimers. a

Scheme 3. Biosynthetic Pathway in S. putrefaciens for E,EPutrebactene (E,E-pbeH2), with the Enzyme Co-factors as Given in Scheme 1

Figure 8. Minimized structures (HyperChem 7.5) of [Fe(pb)]+, [Fe(E-pbx)]+, [Fe(E,E-pbe)]+, [Fe(E,Z-pbe)]+, [Fe(Z-pbx)]+, and [Fe(Z,Z-pbe)]+ viewed along the z-axis, with H atoms and coordinated aqua ligands omitted for clarity.

MS system) across two runs to accommodate the detection of all species. For quantitative analysis, the LC and putrebactin SIM traces were used as internal standards. Fe(III)-loaded samples were prepared by adding 20 μL of 1 mM Fe(III) solution (pH 7) to 80 μL of the siderophore-containing samples, and the solution was left for at least 1 h prior to analysis by LC−MS at 450 nm. Ga(III)-loaded samples were prepared by adding 10 μL of 1 mM Ga(III) solution (pH 7) to 40 μL of the siderophore-containing samples and the solution was left for at least 3 h prior to analysis by LC−MS at 220 nm. Bacterial Culturing. Shewanella putrefaciens ATCC 8071T was obtained from the American Type Culture Collection (ATCC), and permanent stocks were maintained in Difco marine broth 2216 (Bacto) with 10% v/v DMSO at −80 °C. Base medium contained bactopeptone (5 g L−1), yeast extract (2 g L−1) and sea salts (35 g L−1) and was stirred with Chelex 100 resin (10−20 g L−1) for 2 h to remove iron. The pH value of the decanted medium was adjusted to 7.00 ± 0.05 before sterilization with autoclaving (121 °C, 20 min). Milli-Qgrade H2O was used throughout the culturing and purification procedures. All glassware was treated with 0.1% w/v EDTA for 24 h and rinsed with H2O to remove residual iron. Precultures of S. putrefaciens (50 mL) were grown overnight in Fe-depleted medium at RT on a Ratek orbital shaker at 110 rpm. Aliquots (1 mL) of the midlog-phase cell suspension were collected. After centrifugation (4000 rpm, 20 min), the cell pellets were resuspended in fresh medium. The cell suspension was used to inoculate 100-mL cultures of the base medium, which contained 10 mM NaCl. For control cultures no DBO or exogenous diamine substrates were added to the medium. For experiments aimed to reduce endogenous levels of DB, the base medium was augmented prior to inoculation with an aliquot (1 mL) of a 1 M aqueous stock solution of DBO·2HCl. For precursor-directed biosynthesis experiments, the base medium was augmented prior to inoculation with DBO·2HCl, as above; in addition to an aliquot (1 mL) of a 1 M aqueous stock solution of 15N2-DB·2HCl or E-DBE· 2HCl, to give a final total diamine concentration (inhibitor, 10 mM; substrate, 10 mM) of 20 mM. Prior to addition to the medium, the pH value of each diamine stock solution was adjusted using NaOH to pH

Figure 9. Minimized structure (HyperChem 7.5) of (a) [Fe2(pb)3], as generated from the X-ray crystal structure of [Fe2(alcaligin)3]68 and of (b) [Fe2(E,E-pbe)2(E,Z-pbe)]. H atoms have been omitted for clarity. Liquid Chromatography−Mass Spectrometry: Solution Preparation. Aliquots (20 μL) of samples were analyzed as native solutions at 220 nm by LC−MS using positive-ion ESI mode or selected ion monitoring (SIM) detection mode. The SIM detection mode was set for four different target ions (the maximum for the LC− 953



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imported into HyperChem 7.5, and the atoms beyond the first and second coordination sphere subjected to geometry optimization (Polak-Ribiere algorithm; termination condition, RMS gradient 0.2 kJ mol−1). A similar strategy was used to model [Fe2(pb)3] and [Fe2(E,E-pbe)2(E,Z-pbe)].

7.00 ± 0.05 and was filtered using Minisart 0.2 μm syringe filters (Sartorius). Isolation and Purification of Macrocyclic Dihydroxamic Acids: XAD-2 Chromatography. Siderophores were purified from the culture supernatants with some modifications to previous methods.40,41 Bacterial cells were harvested at day 6 after inoculation by centrifugation, and the supernatant was collected and loaded by gravity at a flow rate of 5 mL min−1 onto a column (30 × 2.5 cm i.d., column volume (CV) = 147 cm3) containing XAD-2 resin. The resin had been prepared by batch washing in MeOH (1 CV) and H2O (4 CV) before packing the column and was further equilibrated with H2O after packing. After sample loading, the column was washed with H2O (2 CV), 50% MeOH/H2O (1.5 CV), and 100% MeOH (1.5 CV), and fractions of 20 mL were collected. For siderophore detection, an aliquot of sample (50 μL), H2O (50 μL), CAS assay solution (100 μL), and shuttle solution (4 μL) were mixed in this order, and the absorbance value of the solution was measured at 620 nm after 4 h. The siderophore-positive fractions were eluted in the 50% MeOH/ H2O wash, pooled, and dried in vacuo (external bath ∼38 °C). The dried residue was redissolved in Milli-Q water (5−10 mL), and the sample was filtered through 5,000 molecular weight cutoff (MWCO) centrifugal filter units (volume 20 mL) at 4000 rpm for several 40-min runs using an Eppendorf centrifuge 5810R. The retentate was washed with H2O until all detectable siderophore appeared in the filtrate. The filtrate was then pooled and dried in vacuo (external bath ∼38 °C), and the dried residue was dissolved in a small amount of MeOH (∼3 mL). Insoluble materials were removed by centrifugation (8000 rpm, 5 min) using an Eppendorf centrifuge 5415R. Sephadex LH-20 Chromatography. Samples (1 mL) were loaded onto a column (20 × 1.5 cm i.d., CV = 35 cm3) containing Sephadex LH-20 resin that had been prepared by soaking (∼3 g) in MeOH (50 mL) for 24 h. The column was washed with MeOH at a flow rate of 0.1−0.2 mL min−1, and 1-mL fractions were collected. CAS-positive fractions were air-dried overnight at RT to evaporate MeOH. The dried samples were redissolved in H2O (∼3 mL). C18 Reverse-Phase Chromatography. An aliquot (∼1.5 mL) of the sample was adsorbed onto a single-use prepacked C18 reversephase column (10 × 1 cm i.d.), and the products were eluted using a step gradient of 0−100% MeOH in water (0, 20%, 40%, 60%, 80%, and 100%). Siderophore-containing fractions were collected at 40% MeOH. Ni(II)-Based Immobilized Metal Ion Affinity Chromatography. The IMAC column was manually packed with a 1-mL bed volume of Ni(II) SepharoseTM 6 Fast Flow resin (GE Healthcare). The column was washed with Milli-Q water (5−10 CV) and equilibrated with binding buffer (10 mM HEPES, 0.2 M NaCl, pH 9.0; 5−10 CV). The semipurified siderophore extract (500 μL) was mixed with binding buffer (500 μL) and was loaded onto the column. The column was washed with 5 CV of binding buffer, followed by 5− 10 CV of elution buffer (10 mM HEPES, 0.2 M NaCl, pH 5.5), and fractions of 1 mL were collected. As identified using the CAS assay, the fractions containing siderophore were eluted in the elution buffer, pooled (total volume 3 mL), and lyophilized using a Labconco FreeZone freeze-dryer. To remove HEPES and NaCl, the dried sample was extracted in MeOH (∼ 1 mL), and insoluble materials were removed by centrifugation (12000 rpm, 5 min) using an Eppendorf centrifuge 5415R. The extract was air-dried overnight at RT to evaporate MeOH. The dried sample was redissolved in H2O (∼ 100 μL). Yields. The yields of pbH2, 15N4-pbH2, and E,E-pbeH2 were estimated using the CAS assay, with desferrioxamine B as a reference compound, as 4.2, 11.3, and 2.1 mg L−1, respectively. These estimates included a correction for the trihydroxamic acid (standard)/ dihydroxamic acid (target). Molecular Modeling. Structures of [Fe(pb)(OH2)2]+, [Fe(E,Epbe)(OH2)2]+, [Fe(E-pbx)(OH2)2]+, [Fe(Z,Z-pbe)(OH2)2]+, [Fe(Zpbx)(OH2)2]+, and [Fe(E,Z-pbe)(OH2)2]+ were built using Spartan ’10 using the coordinates of one intraligand-Fe(III) unit of the X-ray crystal structure of Fe2(alcaligin)3 with the two oxygen donor atoms of the bridging ligand replaced with water ligands.68 The structures were

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support from The University of Sydney (Strategic Research Fund, Bridging Grants to R.C.; cofunded postgraduate stipend to C.Z.S.) is gratefully acknowledged.

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ABBREVIATIONS Each diamine inhibitor or substrate has been assigned an uppercase abbreviation: (DBO, 1,4-diamino-2-butanone; DB, 1,4diaminobutane; E-DBE, 1,4-diamino-2(E)-butene). Macrocycles have been assigned a lower-case abbreviation: (pbH2, putrebactin; E,E-pbeH2, E,E-putrebactene; E-pbxH2, E-putrebactene); LC−MS, liquid chromatography−mass spectrometry; ODC, ornithine decarboxylase; SIM, selected ion monitoring

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REFERENCES

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