Structure elucidation and biosynthesis of fuscachelins, peptide siderophores from the moderate thermophile Thermobifida fusca

Abstract

Bacteria belonging to the order Actinomycetales have proven to be an important source of biologically active and often therapeutically useful natural products. The characterization of orphan biosynthetic gene clusters is an emerging and valuable approach to the discovery of novel small molecules. Analysis of the recently sequenced genome of the thermophilic actinomycete Thermobifida fusca revealed an orphan nonribosomal peptide biosynthetic gene cluster coding for an unknown siderophore natural product. T. fusca is a model organism for the study of thermostable cellulases and is a major degrader of plant cell walls. Here, we report the isolation and structure elucidation of the fuscachelins, siderophore natural products produced by T. fusca. In addition, we report the purification and biochemical characterization of the termination module of the nonribosomal peptide synthetase. Biochemical analysis of adenylation domain specificity supports the assignment of this gene cluster as the producer of the fuscachelin siderophores. The proposed nonribosomal peptide biosynthetic pathway exhibits several atypical features, including a macrocyclizing thioesterase that produces a 10-membered cyclic depsipeptide and a nonlinear assembly line, resulting in the unique heterodimeric architecture of the siderophore natural product.

Iron is a nutrient that is required by virtually all organisms to conduct essential life processes. Under aerobic conditions, the ferric oxidation state predominates as the extraordinarily water-insoluble Fe(OH)₃ salt (1). These environmentally limiting conditions have placed selective pressure on organisms to develop controlled and specific mechanisms to acquire iron. Siderophores are secondary metabolites used to scavenge ferric ion selectively through the formation of soluble chelation complexes (2, 3). This structurally diverse group of small molecules contains metal-chelating motifs that commonly include hydroxamates, catechols, α-hydroxyacids, and heterocycles to bind iron with high affinity. Iron uptake is frequently a limiting factor for growth, including in human hosts, making siderophores virulence factors in a variety of human pathogens and a target for antimicrobial therapy (4–6).

The constantly expanding pool of microbial genomic sequence data has prompted the isolation of new natural products through the identification of orphan biosynthetic gene clusters (7–9). Exploiting the predictive nature of biosynthetic pathways, natural products can be isolated and characterized by using an assay-guided fractionation approach. Nonribosomal peptide (NRP) biosynthetic machinery is often used to construct siderophores (10, 11). Peptide-based architectures allow for the incorporation of common iron-chelating functionalities. Structure elucidation of nonribosomal peptide natural products is particularly amenable to a genome mining approach because the assembly-line nature of the enzymatic machinery leads to predictable products. NRP synthetases are large, multidomain enzymes catalyzing the assembly of peptides by a thioester-templated mechanism (10). Identification of amino acid building blocks is possible from analysis of the sequence of the NRP synthetase adenylation domains (12–15). By using this information in combination with the frequently observed colinearity of the biosynthetic genes with the product, prediction of the peptide structure is possible. Despite several recent examples of this approach, there remain aspects of NRP biosynthesis that are difficult to predict, including uncommon amino acid incorporation, domain skipping/repeating, and macrocyclization.

Thermobifida fusca is a moderately thermophilic actinomycete widely studied as a model organism for thermostable extracellular cellulases (16–20). The genomic sequence of T. fusca YX was reported recently (21). There are no characterized secondary metabolites from this actinomycete and few characterized natural products from any thermophilic bacteria or archaea (22). One recent example is the elucidation of benzodiazepine biosynthesis in Streptomyces refuineus (23). Here, we describe a family of structurally novel nonribosomal peptide siderophores, termed fuscachelins, produced by an orphan gene cluster from T. fusca. The elucidated biosynthetic pathway contains many unusual aspects that were not predictable by bioinformatic analysis. In addition, structure elucidation of the fuscachelins revealed a molecular architecture not observed in iron-chelating siderophore secondary metabolites.

Results

Siderophore Gene Cluster in T. fusca.

An uncharacterized gene cluster in T. fusca contains genes corresponding to a multimodular NRP synthetase secondary metabolite biosynthetic pathway [see supporting information (SI) Fig. S1]. Three NRP synthetase genes designated fscGHI are contiguous in the cluster and correspond to five peptide extension modules (Fig. 1A). The first gene, fscG, encodes a 390-kDa protein comprising three NRP synthetase modules, each containing the core elongation domains: condensation (C), adenylation (A), and peptidyl carrier protein (PCP). The first module contains a predicted epimerization (E) domain, suggesting that the stereochemistry of the corresponding amino acid in the product has the D-configuration. The second gene, fscH, contains a single elongation module and is followed by a gene for the termination module, FscI, which contains a C-terminal thioesterase (TE) domain. Upstream of fscGHI are genes with sequence homology to characterized 2,3-dihydroxybenzoic acid (Dhb) biosynthetic genes. FscA, FscB, and FscD are homologous to the well characterized catecholate biosynthetic enzymes: isochorismate synthase, isochorismatase, and 2,3-dihydro-Dhb dehydrogenase (24–26). An adenylation domain (FscC) with predicted specificity for Dhb and a dedicated aryl-carrier protein (FscF) are present as stand-alone domains to incorporate Dhb as the starter building block. Additional proximal genes are present that are homologous to genes traditionally associated with siderophore production and utilization. These include an l-ornithine hydroxylase (FscE), membrane proteins for siderophore export and uptake, and a ferric iron reductase.

Fig. 1.

The predicted gene products of a nonribosomal peptide biosynthetic cluster in T. fusca. (A) Schematic organization of the nonribosomal peptide assembly line components. (C, condensation; A, adenylation; ArCP, aryl carrier protein; E, epimerization; PCP, peptidyl carrier protein; and TE, thioesterase domains). (B) Comparison of the adenylation domain specificity with known nonribosomal peptide natural products (12, 30–34).

The amino acid specificity of the individual modules can be predicted by using methodology that compares active-site residues of known NRP synthetase A domains (Fig. 1B) (27–29). The specificity of the first module of FscG did not correspond convincingly to any characterized domains but suggested activation of a basic amino acid. The second and third modules of FscG are highly similar to each other (≈85% identity over the adenylation domains), and both are predicted to activate glycine. Analysis of FscH and FscI suggests activation of l-serine and l-N^δ-hydroxyornithine (HOOrn), respectively. Based on this analysis, the peptide product can be predicted as a pentapeptide; N-capped with Dhb, a structural architecture unlike any characterized siderophore.

Structure Solution of the T. fusca Siderophores, Fuscachelins.

To resolve the structure of the T. fusca siderophore, the natural product was isolated from the producing bacterium and characterized with NMR and mass spectrometry. T. fusca was grown in iron-depleted Hägerdal medium at 55°C, and the siderophore was extracted from pelleted cells by using methanol. Siderophore activity was monitored throughout production and purification by using the chrome azurol S (CAS) assay for iron-binding activity (35). In addition, fractionation by HPLC was monitored by absorption at 320 nm, characteristic of the predicted catechol functionality. Four separate peaks were isolated from the preparation (Fig. 2A and Fig. S2). A minor peak, with the longest retention time, was the free acid 2,3-Dhb as determined by NMR analysis. A major peak (corresponding to fuscachelin B) was further purified by reverse-phase chromatography, and the structure was determined. ¹H, TOCSY, and COSY experiments established the amino acid content of the peptide by identifying the individual amino acid spin systems: Arg, Gly, Ser, HOOrn in addition to Dhb (Figs. S3 and S4). Unexpectedly, ¹³C and ¹⁵N gHMBC experiments to confirm the amino acid connectivity revealed a heterodimeric peptide with the sequence Dhb-Arg-Gly-Gly-Ser-HOOrn-Gly-Gly-Arg-Dhb (Table 1 and Fig. S5). The mass of the isolated product supports this structure with a measured exact m/z of 1048.4448 ([M+H]⁺, calculated 1048.4448) and a molecular formula of C₄₂H₆₂N₁₅O₁₇ (Fig. S6). Close examination of the ¹H NMR spectra reflects a heterodimeric structure. For example, the integration of the amide and α-C protons is 2:4:1:1, Arg:Gly:Ser:HOOrn (see Fig. S3), and fine double signals are evident in the asymmetric halves of the molecule (for example, the protons of Arg and Dhb). To confirm the NMR structural assignment, MALDI-TOF/TOF fragmentation (Fig. 3 and Fig. S6) was performed, and all fragments are consistent with the predicted structure. The determination of amino acid chirality was conducted by using Marfey's method (36), indicating the presence of d-Arg, Gly, l-Ser, and l-HOOrn in a ≈2:4:1:1 ratio based on peak integration (Fig. S7). An additional chromatographic peak from the T. fusca preparation, eluting just before fuscachelin B, exhibited NMR spectra very similar to fuscachelin B. The measured m/z of this distinct product was 1047.4614 ([M+H]⁺, calculated 1047.4608, C₄₂H₆₃N₁₆O₁₆) corresponding to a change to an NH versus an O in fuscachelin B. Inspection of the ¹H NMR and mass spectral fragmentation data showed that this difference was localized at the HOOrn residue, and additional NMR experiments, in particular ¹H/¹⁵N gHSQC, are consistent with an α-amide structure (termed fuscachelin C) as shown in Fig. 2B (Fig. S8).

Fig. 2.

Purification and structures of fuscachelins A–C. (A) HPLC chromatographic trace illustrates four distinct products from T. fusca containing the catechol functionality. Dhb, dihydroxybenzoic acid. (B) Chemical structures of fuscachelins A–C.

Table 1.

¹H and ¹³C NMR spectral data for fuscachelin B

Position	δH, multiplicity	J, Hz	δC	Position	δH, multiplicity	J, Hz	δC
1			148.3	30	1.61 m		29.1
2			146.1	31	4.28 dd	4.6, 5.3	54.0
3	7.05 d	7.9	121.2	32			176.6
4	6.82 t	8.0	121.2	33	7.93 d	7.7
5	7.24 d	8.1	120.9	34			172.3
6			117.9	35	3.97 m		43.9
7			171.6	36	8.33
8	8.71 d	6.7		37			173.1
9	4.53 dd	7.7	55.3	38	3.95 m		43.7
10	1.98 m		29.5	39	8.78 d
	1.89 m			40		176.3
11	1.71 m		25.9	41	4.53 dd	7.7	55.3
12	3.21 t		42.0	42	1.98 m		29.5
13	7.15 br s				1.89 m
14			158.2	43	1.71 m		25.9
15	6.59 br s			44	3.21 t		42.0
16			176.3	45	7.15 br s
17	8.78 d			46			158.2
18	4.03 m		44.1	47	6.59 br s
19			172.8	48	8.71 d	6.7
20	8.33			49			171.6
21	4.01 m		43.6	50			117.9
22			171.7	51			148.3
23	8.03 d	6.6		52			146.1
24	5.00 dd	5.6, 5.0	54.0	53	7.05 d		121.2
25	3.77 m		62.0	54	6.82 t		121.2
26			171.6	55	7.24 d		120.9
27
28	3.58 m		49.2
	3.50 m
29	1.78 m		25.8

Fig. 3.

MALDI-TOF/TOF fragmentation of fuscachelin B.

A third CAS-positive fraction (fuscachelin A) was evident in the chromatographic separation (see Fig. 1A). This fraction was unstable during the process of purification and appeared to decompose slowly to fuscachelin B. The mass of this peak is 18 Da less than fuscachelin B ([M+H]⁺, m/z predicted: 1030.4342, m/z observed: 1030.4344; see Fig. S6), suggesting a hydration/dehydration process. Aspects of the NMR spectra of fuscachelin A were very similar to those described for fuscachelin B, with the d-Arg and Gly signals remaining largely identical. There were, however, distinct differences in the l-N^δ-hydroxyornithine, l-serine, and Dhb signals. In particular, the β-hydrogens of serine were shifted to 4.48 and 4.36 ppm (compared with 3.77 ppm in fuscachelin B), suggesting the presence of a seryl ester. In addition, shifts in the l-N^δ-hydroxyornithine side-chain δ-protons signals were pronounced. These changes suggested a substantial change in environment of these two central amino acids in the fuscachelins. Furthermore, this change was capable of being communicated to the Dhb moiety in the extremities of the molecule, as is evidenced by the increased complexity of the splitting patterns and broadened peaks of the aromatic protons. Over time, upon standing at ambient temperature (unbuffered D₂O), these changes in proton signals began to disappear as signals for fuscachelin B concurrently grew into the spectrum (Fig. S9). These observations, in concert with the observed mass difference, suggested the presence of a macrolactone involving the serine side chain and the α-carboxyl of l-HOOrn (Fig. 2B). The unusual 10-membered cyclodepsipeptide core structure in fuscachelin A was confirmed by additional NMR-based measurements (Fig. S9). Further evidence for the presence of a macrolactone and the degradation of fuscachelin A to B was provided by the alkali treatment of purified fuscachelin A (Fig. 4). The results show that fuscachelin A is converted to fuscachelin B based on HPLC analysis. As discussed below, the macrolactone fuscachelin A is the most likely initial biosynthetic product of the T. fusca fuscachelin gene cluster. Energy minimization of fuscachelin A results in a structure in which the two catechols are in proximity to the central hydroxamate, and modeling of a metal chelate is easily accommodated (Fig. S10).

Fig. 4.

Proposed origin of fuscachelin B from the product of the biosynthetic gene cluster, fuscachelin A. HPLC analysis showing the proposed hydrolytic conversion of fuscachelin A to fuscachelin B.

Siderophore production and utilization depend on cellular responses to iron in the environment. The concentration of ferrous iron in the growth medium affects the expression of bacterial siderophore biosynthetic genes (1). To provide evidence that the fuscachelins are the biologically relevant siderophore for T. fusca, the bacteria were grown in minimal medium with and without iron, and the production of fuscachelin was monitored by HPLC analysis. As predicted, the presence of iron suppressed fuscachelin production (Fig. S2). In addition, the fuscachelin B–iron complex was prepared and characterized by MALDI-TOF mass spectral analysis to demonstrate that the natural product provides a scaffold for iron binding. The results ([M+Fe]⁺ m/z 1101.3600, C₄₂H₅₉N₁₅O₁₇Fe) agree well with the predicted value of 1101.3557 (Fig. S6).

Cloning, Purification, and Biochemical Characterization of the Terminal Module FscI.

To provide supporting evidence that the fsc genes are responsible for the in vivo production of the fuscachelins in T. fusca, the terminal module, FscI, was characterized in vitro. The 4.0-kb gene for fscI was cloned from T. fusca genomic DNA into an Escherichia coli expression vector using PCR-based methods. The four-domain protein was overexpressed in E. coli to high levels and purified to homogeneity as judged by SDS/PAGE analysis (Fig. 5A). The amino acid specificity of the module was determined by using the pyrophosphate exchange assay of adenylation domain function. l-HOOrn was chemically synthesized by using published protocols from N-Boc-l-ornithine (37). This predicted amino acid was assayed along with the 20 proteinogenic amino acids and l-ornithine. The results show a significant preference for l-HOOrn with the next best amino acid substrate the isosteric l-lysine (Fig. 5B).

Fig. 5.

Characterization of FscI. (A) SDS/polyacrylamide gel illustrating overexpressed and purified FscI (148 kDa). (B) Relative activity of FscI with various amino acids as judged by the pyrophosphate exchange assay for adenylation function.

Discussion

Identifying orphan biosynthetic gene clusters through genome mining has proven to be a successful approach toward the discovery of novel small molecule secondary metabolites. The moderately thermophilic actinomycete, T. fusca, contains one gene cluster belonging to the NRP family of natural products. The cluster contains the hallmarks of siderophore-producing enzymes, with biosynthetic pathways to the iron-chelating 2,3-dihydroxybenzyl and hydroxamide moieties. Although the enzymatic components of the fuscachelin cluster have homologs in other siderophore NRP synthetases, the combination and organization of the fsc genes are unique. Siderophore natural products most often bind to extracellular ferric ions through the formation of a hexacoordinate, octahedral chelation complex. The T. fusca siderophore is able to achieve this through the production of a heterodimeric nonribosomal peptide with two terminal catechols and an internal hydroxamate. The biosynthetic pathway for this siderophore, termed fuscachelin A, is unusual, and prediction of the structure of the natural product would not be possible based on analysis of the genomic sequence alone. Namely, unambiguous assignment of the adenylation domains, prediction of macrocyclization, and the unusual nonlinear biosynthetic pathway were unexpected.

We initially characterized a siderophore natural product (fuscachelin B) from T. fusca as a linear octapeptide N-capped with two 2,3-dihydroxybenzoic acids and an internal α,ε-linked HOOrn. The structure was determined with detailed NMR and mass spectrometric characterization of the isolated product. A second fraction in the purification (fuscachelin C) exhibited characteristics nearly identical to fuscachelin B, with the notable exception of a 1-Da mass difference, corresponding to a change of an oxygen atom to an NH. This change was localized by NMR and mass spectral fragmentation data to the l-HOOrn subunit, specifically as an α-carboxamide group. A third product was isolated from T. fusca that displayed properties similar to those of fuscachelin B and C and slowly decomposed over time, or rapidly with alkali treatment, to fuscachelin B. This product (fuscachelin A) was characterized as a macrolactone, and, based on the predicted biosynthetic pathway, it is most likely the initial natural product of the T. fusca NRP synthetase machinery. We believe that fuscachelins B and C are degradation products of fuscachelin A, resulting from nucleophilic opening of the 10-membered macrolactone: hydrolysis to form fuscachelin B and aminolysis (the growth medium contains 24 mM ammonium sulfate) to form fuscachelin C.

The predicted biosynthetic pathway (Fig. 6) for fuscachelin A contains well characterized genes for the biosynthesis of a peptidic siderophore, although there are several unusual features. The condensation domain of FscI couples both Dhb-Arg-Gly-Gly and Dhb-Arg-Gly-Gly-Ser to the δ-and α-nitrogens of l-HOOrn bound to the PCP domain of FscI. The two coupling partners originate from the carrier domains of FscG and FscH. The second coupling (as diagrammed in Fig. 6) between FscI and FscG involves the skipping of module FscH, an example of nonlinear peptide assembly, observed in a limited number of NRP pathways (38). The coupling of two PCP-bound acyl intermediates to l-HOOrn and the predicted module skipping is reminiscent of the chemistry of the NRP synthetase CchH in coelichelin biosynthesis (12). After the tandem couplings to the HOOrn core, the thioesterase domain of FscI performs a macrocyclization to form the 10-membered depsipeptide ring of fuscachelin A. The FscI TE domain shows significant homology to the thioesterase of DhbF of bacillibactin biosynthesis (30). DhbF catalyzes a related reaction, the cyclotrimerization of Dhb-Ser-Gly to form a 12-membered macrolactone siderophore. To confirm the activity of the gene cluster, fscI was cloned from T. fusca and overexpressed in E. coli. The adenylation domain of the purified protein was biochemically demonstrated to prefer the nonproteinogenic amino acid l-HOOrn. This result both establishes the role and position of the NRP synthetase FscI in the pathway and demonstrates that the flavin monooxygenase (FscE) acts on the free amino acid l-ornithine and not the mature peptide.

Fig. 6.

Proposed biosynthetic pathway to fuscachelin A.

Here, we present the structure elucidation of a siderophore from the thermophilic actinomycete T. fusca. The natural product was discovered by using a genome-mining approach. Fuscachelin A is the first secondary metabolite isolated from this bacterium, and the described natural product gene cluster is one of only a few from any thermophilic species. The proposed biosynthetic pathway contains unusual aspects that demonstrate the flexibility of NRP assembly-line chemistry to construct biologically active peptides, and the structure of fuscachelin A represents a new molecular architecture for the chelation of iron.

Materials and Methods

Purification of Fuscachelins A–C.

T. fusca spores (ATCC 2773) were used to inoculate 5-ml liquid cultures grown in LB broth at 55°C and 150 rpm in a Thermo Electron Corporation Forma Orbital Shaker. After 48 h, the cells were thoroughly exchanged into 5 ml of iron-deficient Hägerdal medium (39) by repeated centrifugation and used to inoculate 1-liter liquid cultures, also in iron-deficient Hägerdal medium. One-liter cultures were grown for 7 days at 55°C and 150 rpm in a Thermo Electron Corporation Forma Orbital Shaker. Cells were pelleted at 10,000 rpm and 4°C for 30 min by using a Beckman–Coulter J2-HS centrifuge. Cell pellets were collected and extracted five times with 30-ml portions of methanol. The methanol extracts were concentrated in vacuo to a small volume (≈5 ml), and siderophore activity was confirmed by using the CAS assay of Schwyn and Neilands (35). CAS-positive extracts were concentrated to dryness and subjected to two rounds of preparative HPLC by using a Vydac 218TP1022 protein and peptide C18 column (250 × 22 mm, 10 μm) on a Shimadzu LC-6AD liquid chromatography system equipped with a Shimadzu SPD-10A UV-visible detector. A detection wavelength of 320 nm was chosen to monitor elution of the 2,3-dihydroxybenzyl moiety. A linear gradient of 2–50% methanol in 0.1% trifluoroacetic acid and water was run over 30 min at 10 ml min⁻¹. CAS-positive fractions were collected between 23 and 28 min and lyophilized. The lyophilized material was dissolved in 5 ml of 23% methanol and water. One-milliliter portions were injected onto the column and purified by using a linear gradient of 23–33% methanol in water and 0.1% trifluoroacetic acid over 60 min at 8 ml min⁻¹. Three CAS-positive fractions were collected at 17.1, 18.6, and 22.8 min. Lyophilization of the CAS-positive fractions afforded 2.0, 3.0, and 2.3 mg of fuscachelins C, B, and A, respectively, as white solids. The fourth CAS-positive fraction (eluting at 24.8 min) was free 2,3-dihydroxybenzoic acid as judged by ¹H NMR. Samples for ¹⁵N NMR analysis were prepared in an identical fashion with the exception that (¹⁵NH₄)₂SO₄ (99% ¹⁵N, Cambridge Isotope Laboratories) was included in the Hägerdal medium as the sole nitrogen source.

NMR Structure Elucidation.

All NMR spectra were acquired by using a D₂O susceptibility matched 5-mm Shigemi advanced NMR microtube. NMR solvents were purchased from Cambridge Isotope Laboratories. ¹H, ¹³C, ¹⁵N, and ¹H-¹³C correlation spectra were acquired on a Varian INOVA 500 MHz (¹H) NMR spectrometer with VNMR 6.1C software. Spectra were processed by using MestReC version 4.9.9.8 software. Spectra acquired in D₂O were referenced to the residual HOD peak. In cases where an H₂O/D₂O (4:1) solvent mixture was used to observe backbone amide protons and exchangeable side chain protons, the intense solvent peak was suppressed by using the PRESAT and solvent suppression parameters in the VNMR 6.1C software. ¹H, TOCSY (512 increments, 64 transients, 80-ms mixing time), gCOSY (512 increments, 48 transients), gHSQC (512 increments, 32 transients), and gHMBC (512 increments, 64 transients) spectra were acquired by using a Varian 500 ID/PFG 50-202-MHz inverse probe. ¹³C and ¹⁵N spectra were acquired by using a Varian 500 SW/PFG 50-202-MHz broadband probe. ¹³C spectra were referenced to CD₃OD, which was used as an internal standard. ¹⁵N spectra were externally referenced to 2.9 M ¹⁵NH₄Cl/1 M DCl in D₂O (Cambridge Isotope Laboratories). ¹H-¹⁵N correlation spectra (gHSQC, 256 increments, 4 transients; and gHMBC, 128 increments, 16 transients) were acquired on a Varian VNMRS 600-MHz (¹H) NMR spectrometer equipped with a 5-mm HCN AutoX inverse probe.

MS Analysis.

High-resolution ESI+ single-mass analysis was performed on an Agilent LC/MSD TOF mass spectrometer equipped with Agilent Technologies 1100 series cap-LC pumps. MS/MS experiments were performed on an Applied Biosystems/MDS SCIEX 4800 MALDI-TOF/TOF mass spectrometer. A ferric–fuscachelin B complex was prepared by mixing purified fuscachelin B with 1% FeCl₃ in a 1:1 siderophore:Fe ratio (40). The solution was mixed at room temperature for 2 h and lyophilized. The dried sample was subjected to ESI+ analysis by using a Micromass LCT-TOF mass spectrometer.

Marfey's Method Analysis of Amino Acids.

Five hundred micrograms of fuscachelin B was dissolved in 400 μl of 6 M HCl and heated at 110°C for 24 h. The mixture was lyophilized, and the residue was dissolved in 10 μl of water, 20 μl of 1 M NaHCO₃, and 170 μl of 1% Marfey's reagent [N^α-(2,4-dinitro-5-fluorophenyl)-l-alaninamide, Sigma–Aldrich] acetone solution and heated at 37°C for 1 h. The reaction was quenched with 20 μl of 1 M HCl and the mixture lyophilized. The dried products were dissolved in 1:1 water:acetonitrile and 0.1% trifluoroacetic acid solution to a final volume of 400 μl and analyzed on a Vydac 218TP54 protein and peptide C18 HPLC column (250 × 4.6 mm, 5 μm) on a Shimadzu LC-6AD liquid chromatography system equipped with a Shimadzu SPD-10A UV-visible detector. The separation was performed by using a linear gradient of 0- 52.5% buffer B (10 mM ammonium formate, 1% methanol, 60% acetonitrile, pH 5.2) in buffer A (10 mM ammonium formate, 1% methanol, 5% acetonitrile, pH 5.2) over 45 min by using detection wavelengths of 340 and 220 nm, an injection volume of 5 μl, and a volume flow rate of 1 ml min⁻¹ (36). Stereochemical assignments were made by comparison with the retention times of Marfey's derivatives prepared from authentic amino acid standards of d/l-arginine, d/l-serine, d/l-ornithine, and glycine (Sigma–Aldrich). In addition, the peak for l-HOOrn was identified by using the Marfey's derivative of a synthetic standard of l-HOOrn that was chemically synthesized by using an established protocol (37). Marfey's derivatives of HPLC amino acid standards were prepared by mixing 50 μl of 50 mM (aq) amino acid, 100 μl of 1% Marfey's reagent/acetone solution, and 20 μl of 1 M NaHCO₃, then heating at 37°C for 1 h, lyophilizing, and dissolving in 1:1 water:acetonitrile and 0.1% trifluoroacetic acid solution to a final volume of 400 μl. Separation of the standard mixture was optimized to the conditions outlined above.

Fuscachelin A HPLC Peak Shift Analysis.

Twenty microliters of 100 μM standards of purified fuscachelins A and B were run on a Vydac 218TP54 protein and peptide C18 HPLC column (250 × 4.6 mm, 5 μm) by using a linear gradient of 23–28% B (methanol) in A (water and 0.1% trifluoroacetic acid) over 30 min at 1 ml min⁻¹. HPLC traces were acquired at 220 and 335 nm. Then, a 300-μl sample of 100 μM fuscachelin A in 100 mM phosphate buffer (pH 10) was shaken at 37°C for 2 h. The reaction mixture was lyophilized to dryness, taken up in 23% methanol and water to a final volume of 300 μl, and subjected to the HPLC conditions outlined above.

Cloning, Expression, and Purification of FscI.

The gene for fscI was amplified by using PCR from T. fusca genomic DNA with the following primers: 5′-GCG GAA TTC ACC ACC GCA GCC GCG GGT (EcoRI), 5′-GCG AAG CTT CTA GCT GTG TCC GGA TCG (HindIII). The PCR products were purified through agarose gel electrophoresis and gel extraction (Qiagen) and cleaved with the EcoRI and HindIII restriction endonucleases. The fscI gene was then ligated into the plasmid pET30a. The plasmid was transformed into E. coli BL21(DE3) cells for gene expression. Cultures (1 liter) were grown to A₆₀₀ = 0.5–0.7 at 37°C, at which point the shaker was cooled to 18°C, and overexpression was initiated by the addition of 50 μM IPTG. Cultures were continued for 18 h and were harvested by centrifugation, followed by resuspension in 500 mM NaCl, 20 mM Tris·HCl (pH 7.5) and lysed by passage through a French pressure cell at 1,000 psi. Lysate was centrifuged at 10,000 rpm for 20 min in a Beckman Coulter J2-HS centrifuge. The supernatant was incubated for 1 h with 1 ml of metal-affinity resin (Talon resin; Clontech). Resin was washed with 4 × 10 ml of 500 mM NaCl, 20 mM Tris·HCl (pH 7.5), and protein was eluted with 2 × 10 ml of 500 mM NaCl, 20 mM Tris·HCl (pH 7.5), 250 mM imidazole. Protein was dialyzed against 100 mM NaCl, 20 mM Tris·HCl (pH 7.5), 1 mM β-mercaptoethanol, 10% (vol/vol) glycerol, concentrated to 17 μM, and flash frozen.

Pyrophosphate Exchange Assay.

Amino acid-dependent ATP-sodium pyrophosphate assays were performed as follows. A 100-μl reaction contained 75 mM Tris·HCl (pH 8.0), 10 mM MgCl₂, 5 mM DTT, 5 mM ATP, 1 mM Na₄³²P₂O₇, 100 μg/ml⁻¹ BSA, 1 mM amino acid, 2 μM FscI. Reactions were initiated by addition of enzyme and incubated at 30°C for 0.5 h. The reaction was quenched by the addition of 500 μl of 3.5% charcoal, 1.6% perchloric acid, 200 mM Na₄P₂O₇. The charcoal was centrifuged and resuspended twice with 500 μl of 1.6% perchloric acid, 200 mM Na₄P₂O₇. After washing, the charcoal was mixed with 3 ml of scintillation fluid and read by a Beckman–Coulter LS 6500 scintillation counter. All reactions were performed in triplicate.