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Applied and Environmental Microbiology, February 2004, p. 831-836, Vol. 70, No. 2
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.2.831-836.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Degradation Pathway and Generation of Monohydroxamic Acids from the Trihydroxamate Siderophore Deferrioxamine B

Agnes Pierwola,1 Tomasz Krupinski,1 Peter Zalupski,2 Michael Chiarelli,2 and Domenic Castignetti1*

Departments of Biology,1 Chemistry, Loyola University of Chicago, Chicago, Illinois 606262

Received 13 June 2003/ Accepted 23 October 2003


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Siderophores are avid ferric ion-chelating molecules that sequester the metal for microbes. Microbes elicit siderophores in numerous and different environments, but the means by which these molecules reenter the carbon and nitrogen cycles is poorly understood. The metabolism of the trihydroxamic acid siderophore deferrioxamine B by a Mesorhizobium loti isolated from soil was investigated. Specifically, the pathway by which the compound is cleaved into its constituent monohydroxamates was examined. High-performance liquid chromatography and mass-spectroscopy analyses demonstrated that M. loti enzyme preparations degraded deferrioxamine B, yielding a mass-to-charge (m/z) 361 dihydroxamic acid intermediate and an m/z 219 monohydroxamate. The dihydroxamic acid was further degraded to yield a second molecule of the m/z 219 monohydroxamate as well as an m/z 161 monohydroxamate. These studies indicate that the dissimilation of deferrioxamine B by M. loti proceeds by a specific, achiral degradation and likely represents the reversal by which hydroxamate siderophores are thought to be synthesized.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Elemental iron (Fe) is required to synthesize a number of key enzymes and biomolecules (1, 8, 14, 24-26). Iron's biological availability, however, is restricted in those environments where oxygen gas is present and the prevailing pH is near neutrality or is alkaline. As the solution to the problem of acquiring sufficient amounts of Fe, numerous microbes employ siderophores, that is, avid, organic, microbial ferric ion chelators which sequester iron from environments where it is in short supply. Siderophores are thus essential to the nutrition of microbes existing in environments that would otherwise limit their growth (1, 8, 14, 24, 25). Such environments include fresh and marine waters, divergent soils, and living organisms (8, 10, 14, 16, 27-29). In these ecosystems, micromolar concentrations of siderophores have been noted (16, 27, 28).

Siderophores are virulence factors for both animal and plant pathogens (2, 3, 9-11, 18, 23). Indeed, the sequestration of iron by the host is an innate immune mechanism that may limit the course of pathogenic infections (8, 14). Much research has been conducted to investigate the biosynthesis, iron chelation, iron assimilation, and genetics which allow microbes to acquire iron via siderophores. Far less attention, however, has been given to the study of how siderophores are mineralized and returned to the carbon and nitrogen cycles.

Three siderophore-degrading microbes, a pseudomonad (33-35), Azospirillum irakense (36, 37), and Mesorhizobium loti (4, 7, 17, 39), catabolize siderophores concomitant with their growth. Neilands and colleagues (33-35) observed that their microbe, named Pseudomonas FC1, degraded the hydroxamate siderophores ferrichrome, ferrichrome A, and coprogen, with ferrichrome A being the most facile to degrade. Pseudomonas FC1 siderophore degradation was due to inducible enzymes and could occur with either the deferrisiderophore or the ferrisiderophore. The enzymes required to degrade ferrichrome A were cellular, and ferrichrome A was assimilated prior to its being degraded. Potential ferrichrome A degradation products, hypothesized from the structure of ferrichrome A (trans-B-methylglutaconic acid, acetate, and glutamine), supported the bacterium's growth when supplied as nutrients, and with catabolism, ferrichrome A mineralization resulted in the release of monohydroxamates to the culture medium. These authors noted that the enzyme responsible for ferrichrome A degradation was an alkaline protease capable of degrading small, cyclic peptides. With use of enriched alkaline protease preparations, data were presented that indicated a stepwise dissimilation of the cyclic trihydroxamate (ferrichrome A) to a corresponding dihydroxamate and a monohydroxamate followed by dissimilation of the dihydroxamate to monohydroxamates. The ferrichrome A-degrading enzyme, however, was devoid of general amidase (peptidase) activity, and the monohydroxamates generated were hypothesized to subsequently undergo reduction to the corresponding amides, followed by deacylation, such that the products that were released entered intermediary metabolite pools (33, 35).

A. irakense, isolated from lake water, is a nitrogen fixer and degrades the trihydroxamic acid siderophores deferrioxamines B (DFB) and E (36, 37). Similar to that of Pseudomonas FC1, the bacterium's ability to degrade siderophores is inducible, and the enzyme(s) responsible for initiation of siderophore degradation is cellular. The cognate ferrisiderophores, ferrioxamines B and E, are not well degraded but do function as ferric ion sources. The catabolism of DFB occurs via amide hydrolysis. As with Pseudomonas FC1, the degradation of siderophores is limited (desferricoprogen and desferrichrysin are not degraded), and cells of the A. irakense isolate release dihydroxamates and monohydroxamates into the culture medium. A partially purified preparation of the enzyme(s) responsible for DFB hydrolysis yielded dihydroxamates and monohydroxamates, and the enzyme(s) functions with an achiral, nonpeptidic substrate, since amino acid side chains were not required for catalysis (36).

Using high-performance liquid chromatography (HPLC) and positive ion mass spectrometry, Winkelmann and colleagues (36) presented evidence that the trihydroxamate DFB (m/z 561) was degraded to a monohydroxamate of m/z 219 and an N-hydroxy-monohydroxamate of 319. Additional data demonstrated the presence of two distinct dihydroxamates, with m/z of 361 and m/z 419. A presented pathway of DFB degradation indicated its hydrolysis to dihydroxamates (m/z 361 and 419) and monohydroxamates (m/z 219 and 161) resulting from attack at DFB's two amide bonds. Further degradation of the m/z 361 dihydroxamate at its hydroxamate bond generated the m/z 319 N-hydroxy-monohydroxamate and the putative hydrolysis product, acetic acid (36).

M. loti is similar to A. irakense in that it grows and reproduces using DFB as its sole carbon source (4). Unlike for the A. irakense isolate, however, the ferric analogue of DFB, ferrioxamine B (FB), serves as neither an iron source nor a carbon source for M. loti (7). M. loti also differs from A. irakense in that no DFB degradation products are released to the culture medium and such products were noted only when a cell extract, containing the cytosolic, DFB-degrading enzyme(s) (trivially named DFB hydrolase [4, 5, 17, 39]) was given DFB as a substrate.

Using a colorimetric assay (5) that differentiates trihydroxamates from monohydroxamates, the DFB hydrolase from the M. loti isolate generated monohydroxamate(s), and potentially a dihydroxamate(s), from the parent trihydroxamic acid substrate. Since DFB is an asymmetrical molecule, its degradation may proceed with the hydrolysis of one of its two amide bonds or may occur via hydrolysis of the molecule at both amide bonds (Fig. 1). Whether M. loti's DFB hydrolase produced the same or different DFB degradation products as the A. irakense isolate is unknown and was the focus of the present investigation, as was the determination of the pathway and products of DFB degradation by the microbe's DFB hydrolase.



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  		FIG. 1. Hypothesized pathways of DFB degradation to monohydroxamates by the DFB hydrolase preparation. Mass to charge ratios of DFB and the hypothesized intermediates and products would be increased by 1 to represent the additional H+ of these ions. Angled arrows represent proposed sites of initial hydrolysis. Compound I, DFB; Compound II, MH1; Compound III, MH2; Compound IV, DH.

 

   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Growth conditions and cell harvesting.
M. loti was aerobically grown in a mineral salts medium supplemented with 5 ml of vitamin solution liter-1 and 0.1 to 0.3% of DFB as described previously (4, 17, 39). DFB was supplied as mesylate salt by Novartis Pharmaceuticals Corporation (Summit, N.J.). DFB hydrolase was harvested from M. loti as previously described (17, 39). Briefly, late-logarithmic-phase cells were harvested, and cell-free enzyme extract was obtained via sonication to approximately 90% breakage. The cell extract was then subjected to centrifugation (10,000 x g for 10 min to remove cells and large pieces of cell debris) followed by an additional centrifugation (140,000 x g for 90 min) that produced the soluble fraction. Salting out with ammonium sulfate (25 to 45%) was next performed as described previously (4, 39). The DFB hydrolase-containing precipitate of the last step was resolubilized in 5 ml of 50 mM (pH 7.0) phosphate buffer to a protein concentration of about 10.6 mg ml-1.

Preparation of degradation fragments.
To prepare the initial degradation mixture, 0.3 ml of the enriched DFB hydrolase preparation was added to 0.48 ml of 10 mM DFB and 1.46 ml of 50 mM (pH 7.0) phosphate buffer, and incubations were at room temperature (22°C). Aliquots of this reaction mixture were removed, and DFB degradation was stopped at 0, 40, 45, 90, and 120 min and 24 h by lowering the pH to 5 to 6 with 1N HCl, typically by removing 0.6 ml of the enzyme-DFB mixture and adding 2.0 ml of distilled, deionized water and 0.6 ml of 0.1 M HCl. Three negative controls were performed: (i) boiled enzyme, (ii) no DFB (enzyme without substrate), and (iii) no enzyme (substrate without enzyme). The last two controls were performed at each time point, and the equal volume of 50 mM (pH 7.0) phosphate buffer was substituted for the volumes of the missing reactants. All experiments were repeated at least twice to ensure reproducibility. Prior to the injection into the HPLC, direct mass spectrometry (MS), or liquid chromatography-mass spectrometry (LC/MS), samples were passed though 3,000-nominal-molecular-weight-cutoff (NMWCO) Microcon centrifugal filters (Millipore, Bedford, Mass.) which had been spin rinsed with deionized H2O.

HPLC.
DFB degradation fragments were separated using an acetonitrile-water gradient method (36) modified to obtain increased resolution of these compounds. A reverse-phase column (symmetry C18, 5 µm, 3.9 by 150 mm; Waters, Milford, Mass.) was connected to a Waters 1525 binary HPLC pump system equipped with a Waters 717 Plus autosampler, a 2487 dual {lambda} absorbance detector, and a Fraction Collector II (Waters). Detector wavelengths were set at 220 and 435 nm. Except for trifluoroacetic acid (TFA) (Aldrich Chemical Co., Milwaukee, Wis.) and purified, distilled, deionized water (MilliQ system; Millipore), all reagents were purchased from Fisher Scientific (Hanover Park, Ill.). All solvents were degassed and filtered through 0.45-µm-pore-size membrane filters (Gelman Sciences, Ann Arbor, Mich.). The elution gradient used consisted of a combined flow rate of 1 ml min-1 of 97% A (water containing 0.1% TFA) and 3% B (acetonitrile containing 0.1% TFA) for 5 min, which then decreased to 80% A and 20% B for the following 20 min. Over the next 3 min (min 25 to 28), the percentage of A declined to 0 and the percentage of B increased to 100, where it remained for 5 min (min 28 to 33). From 33 to 35 min, the percent A increased from 0 to 97 while the percent B declined from 100 to 3. The last 5 min (min 35 to 40) maintained these percentages prior to the injection of another sample. DFB and all of the degradation fragments eluted by the time a 20% acetonitrile concentration had been obtained. When samples were not eluted from the column for further mass analysis, TFA was used as an ion-pairing reagent to improve the resolution and peak shape. Formic acid was substituted for TFA during the preparation of samples for mass-spectrometric analysis in order to minimize the signal suppression caused by TFA. The gradient profile used when working with formic acid consisted of a combined flow rate of 1 ml min-1 of 100% A (water containing 0.1% formic acid) and 0% B (acetonitrile containing 0.1% formic acid) for 5 min, which then decreased to 80% A and 20% B for the following 20 min. Over the next 3 min (min 25 to 28), the percent A declined to 0 and the percent B increased to 100, where it remained for 5 min (min 28 to 33). From 33 to 35 min, the percent A increased from 0 to 100 while the percent B declined from 100 to 0. The last 5 min (min 35 to 40) maintained these percentages prior to the injection of another sample. The samples were analyzed by direct injection- or liquid chromatography-mass spectrometry either directly after preparation (1 to 2 h) or, when having been frozen at -80°C for up to 1 week, immediately after thawing.

LC/MS.
All solvents and reagents were purchased from Fisher Scientific. Sample components were analyzed using a C18 capillary column (Magic C18; 5 µm, 300 Å, 0.3 by 150 mm; Micromass Bioresources, San Diego, Calif.). A solvent delivery system (ThermoSeparation Products, Riviera Beach, Fla.) operated at a minimal flow rate of 400 µl min-1, which was reduced to 4 µl/min using a flow splitter (Accurate; LC Packings, San Francisco, Calif.). The gradient program and solvents were exactly the same as described above for the HPLC analysis. All samples were loaded onto a column using a 2-µl sample loop.

Mass spectrometry.
The LCQ Advantage (ThermoFinnigan, San Jose, Calif.) quadrupole ion trap was the mass analyzer. The instrument was operated in a positive ion mode. The spray voltage was 3.8 kV, capillary temperature was 200°C, and sheath gas flow rate was 20 liter/min. Tandem mass spectrometry (MS/MS) was conducted on specified parent ions using helium for fragmentation. The fragmentation energy was 30 V. Direct sample injections bypassed the column, and isocratic mobile phase conditions were used (50:50, A/B). Similar conditions have been used to successfully analyze FB and its MS/MS products (13).

Assays.
The presence of DFB and its degradation products was assessed by a spectral assay based on the shifts in absorption maxima of tri-, di- and monohydroxamates (5). This assay was used to determine whether DFB degradation was occurring prior to loading samples for HPLC, MS or LC/MS. Protein concentrations were estimated using the bicinchonic acid procedure (Pierce, Rockford, Ill.).


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Initial attempts to obtain the hypothesized DFB degradation products were performed using HPLC with 0.1% TFA as the ion pair agent. Previous researchers (36) developed a separation technique, but our use of their parameters resulted in insufficient separation of peaks. Increasing the elution time to reach 20% acetonitrile resulted in the separation of the purported monohydroxamate 1 (MH1) (Fig. 1, compound II) at approximately 4 min, monohydroxamate 2 (MH2) (Fig. 1, compound III) at approximately 6 min, dihydroxamate (DH) (Fig. 1, compound IV) at about 18 min, DFB at 22 min, and FB, the DFB Fe adduct, at approximately 16 min, although this peak was often too small to be adequately observed. When formic acid was substituted for TFA as the ion pair agent, the elution order of the purported compounds was the same but the retention times changed. MH1 eluted at approximately 3 min, MH2 eluted at approximately 4.5 min, dihydroxamate eluted at about 16 min, DFB eluted at 20 min and FB eluted at approximately 14.5 min (data not shown). Over a 2-h period, the DFB hydrolase reactions resulted in essentially complete use of DFB and the purported dihydroxamate generated from it, with the final products being the hypothesized MH1 and MH2 (data not shown).

To determine the mass and to deduce the structures of the purported dihydroxamate and monohydroxamates, samples containing mixtures of DFB and its degradation products were directly injected into the MS. Samples taken after 0, 45, and 90 min of DFB degradation by the DFB hydrolase preparation yielded data (Fig. 2 and Table 1) that showed a progressive diminution of the substrate (DFB; m/z 561) concomitant with the synthesis and consumption of the m/z 361 dihydroxamate and the synthesis of the ultimate products, the monohydroxamates MH1 (m/z 161) and MH2 (m/z 219). The results of Table 1 were consistent with either samples injected into the LC/MS or individual fractions separated and collected via the Waters HPLC and then individually injected into the MS. The latter experiments confirmed that the peaks previously observed via HPLC separation as the putative MH1, MH2, DH, DFB, and FB peaks had m/z values of 161, 219, 361, 561, and 614, respectively (data not shown).



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  		FIG. 2. Electrospray mass spectra (positive mode) of DFB after 45 min of degradation by the DFB hydrolase preparation. The sample had been incubated for 45 min prior to being acidified, passed through a 3,000-NMWCO filter, and then injected into the MS.

 

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  		TABLE 1. Electrospray mass spectroscopy detection of DFB and its degradation products (FB [m/z 614.5], DFB [m/z 561.5], DH [m/z 361.3], MH2 [m/z 219.2], and MH1 [m/z 161.2]) observed during 90 min of incubation with the DFB hydrolase preparationa

 
To confirm that the m/z values of these ions were genuine, collision-induced dissociations of the ions were performed. Fragmentation patterns of each of the individual ions listed above were consistent, with the same fragmentation pattern being observed on two or more different occasions (data not shown). Further, controls, such as either no DFB hydrolase (Table 1), heat-inactivated (100°C for 10 min) DFB hydrolase, and no DFB, all confirmed that the generation of MH1, MH2, and the DH occurred only when DFB was subjected to degradation by the DFB hydrolase preparation and that DFB was chemically stable for extended periods of time, up to 24 h.


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Siderophore synthesis is accomplished via nonribosomal peptide synthetases that constitute coordinated, multimodular enzymes which sequentially process intermediates in an "assembly line" fashion (6, 30, 38). Four types of processes are accomplished by the multimodular enzymes: (i) peptidyl carrier proteins contain a phosphopantetheinyl attachment that allows the biosynthetic intermediates to be tethered to the multimodular enzymes; (ii) the processing of intermediates begins with the selection and activation of monomeric substrates or intermediates; (iii) siderophore subunits are incorporated into the siderophore via chain elongation mechanisms, and 4-chain termination releases the siderophore from the multimodular complex (6, 30). For the hydroxamate siderophore rhizobactin 1021, the proposed pathway of biosynthesis involves the generation of 1,3-diaminopropane from glutamic acid and aspartic-ß-semialdehyde (20). One of the nitrogens of the 1,3-diaminopropane is N hydroxylated to generate the corresponding hydroxylamino compound, N4-hydroxy-1-aminopropane. The constituent monohydroxamic acid of rhizobactin 1021 is made once the N4-hydroxy-1-aminopropane is acylated via reacting with acetyl-coenzyme A to yield N4-acetyl-N4-hydroxy-1-aminopropane. Two molecules of N4-acetyl-N4-hydroxy-1-aminopropane are condensed with citric acid to yield the functional base of the siderophore. Addition of the characteristic lipid of rhizobactin 1021 to the acetyl end of one of the N4-acetyl-N4-hydroxy-1-aminopropane subunits completes the synthesis of the siderophore (20).

The DFB biosynthetic pathway and the complement of enzymes responsible have not been fully elucidated (10). Evidence suggests, however, that at least two enzymes exist that perform functions similar to those expected in rhizobactin 1021 synthesis, namely, a lysine decarboxylase, to yield cadaverine, and an oxygenase, which N hydroxylates one of the N atoms of cadaverine (10, 15, 31, 32). As with rhizobactin 1021, a reasonable hypothesis of DFB synthesis stems from the generation of its characteristic monohydroxamates [two molecules of 3-(5'-aminopentyl)-N-hydroxycarbamoylpropanoic acid and one molecule of N-(5-aminopentyl)-N-hydroxyacetamide] followed by their condensation to yield the trihydroxamate siderophore DFB.

M. loti's degradation of DFB to its constituent monohydroxamates is the apparent reversal of the latter condensation steps of the hypothesized biosynthetic pathway. DFB degradation by DFB hydrolase generated only the DH (m/z 361), MH1 (m/z 161), and MH2 (m/z 219), indicating that the pathway depicted in Fig. 1B is operational. The M. loti DFB hydrolase asymmetrically hydrolyzes its substrate; only the m/z 361 DH was observed, and in no case was the hypothesized m/z 419 DH of Fig. 1A noted. This is in contrast to the observations of Winkelmann et al. (36), who reported the synthesis of both the m/z 419 DH and an m/z 319 monohydroxamate.

Analysis of the kinetic data of HPLC and MS also supports the pathway depicted in Fig. 1B, since MH2 (m/z 219) preceded the appearance of MH1 (m/z 161). This is as expected if the latter monohydroxamate (MH1; m/z 161) was formed only after the generation of DH (m/z 361), while the synthesis of MH2 (m/z 219) is concurrent with that of DH.

The earlier study of Winkelmann et al. (36) noted that DFB degradation by A. irakense resulted in some of the same metabolites (DH [m/z 361] and MH2 [(m/z 219]) as those noted in the present study using M. loti as the enzyme(s) source. A. irakense, however, also produced an N-hydroxy-monohydroxamate (m/z 319) and the dihydroxamate of m/z 419, although the latter compound was reported as a minor product. The generation of the N-hydroxy-monohydroxamate (m/z 319) by the A. irakense DFB hydrolase is noteworthy, since it represents the liberation of acetate from the m/z 361 DH prior to the DH being hydrolyzed to the corresponding monohydroxamate. Compared to M. loti, why A. irakense synthesized these additional products (the m/z 419 dihydroxamate and the m/z 319 N-hydroxy-monohydroxamate) is unknown. Clarification may result once the purification to homogeneity of DFB hydrolase from both microbes is achieved.

The rhizobia are known as symbiotic nitrogen fixers (21), yet the ability of rhizobia to degrade unusual compounds has been recognized (12, 22). The rhizobia may thus have ecological roles pertaining to soil nutrient cycling outside of their N fixation capacities (12, 22). Catechols (of interest, since a major class of siderophores is catechol based) are also degraded and, in some cases, used as sole sources of C for growth by the rhizobia (12). M. loti’s ability to mineralize DFB, a siderophore produced by the soil microbe Streptomyces pilosus and other soil Streptomyces spp. (10, 31, 32, 36, 37), is another such example.

Intact trihydroxamic acid siderophores either are not used or are poorly used as sources of Fe by plants (19). In contrast, however, the trihydroxamic acid degradation products (dihydroxamates and monohydroxamates) readily supply Fe to the plant (19). Since plant roots are likely colonized by numerous siderophore-producing prokaryotes and eukaryotes (14, 19, 27-29), the degradation of these molecules to simpler di- and monohydroxamates would help to explain how plants acquire sufficient amounts of Fe even when siderophore-producing microbes exist in the rhizosphere. Whether the release of DFB hydrolase from the M. loti isolate or the release of similar enzymes from currently uncharacterized siderophore-degrading microbes occurs and promotes this process awaits further elucidation.

Our working hypothesis is that the monohydroxamates generated by the DFB hydrolase preparation are most likely reduced to the corresponding amides and then deacylated to yield cadaverine and succinic and acetic acids (17). Both cadaverine and acetic acid support the growth of the M. loti (17), and succinic acid is likely used, since M. loti is an obligate, respiratory bacterium and hence should possess a Krebs cycle. Plans for future study include the determination of whether such products are synthesized by M. loti as well as characterizing and purifying to homogeneity the DFB hydrolase. Additional goals will be to identify, clone, and sequence the DFB hydrolase gene(s) and, since M. loti does not use FB as an Fe source, to isolate and identify the siderophore of the bacterium that allows it to acquire the Fe it needs when the element is in short supply, as occurs when the microbe utilizes DFB as its sole carbon source and the DFB in the medium far exceeds the amount of Fe (as FB) present (4, 17).


   ACKNOWLEDGMENTS
 
This work was supported in part by USDA grants NRI 2002-35101-11532 and NRI 2003-35107-13886 and a Loyola University research support grant to D.C. and by NSF grant DUE 0088566 to M.C.


   FOOTNOTES
 
* Corresponding author. Mailing address: Department of Biology, Loyola University of Chicago, Damen Hall, 6525 N. Sheridan Rd., Chicago, IL 60626. Phone: (773) 508-3638. Fax: (773) 508-3646. E-mail: dcastig{at}luc.edu. Back


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Applied and Environmental Microbiology, February 2004, p. 831-836, Vol. 70, No. 2
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.2.831-836.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.




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