Biosynthetic tailoring of microcin E492m: Post-translational modification affords an antibacterial siderophore-peptide conjugate

Article abstract of DOI:10.1021/ja074650f

The present work reveals that four proteins, MceCDIJ, encoded by the MccE492 gene cluster are responsible for the remarkable post-translational tailoring of microcin E492 (MccE492), an 84-residue protein toxin secreted by Klebsiella pneumonaie RYC492 that targets neighboring Gram-negative species. This modification results in attachment of a linearized and monoglycosylated derivative of enterobactin, a nonribosomal peptide and iron scavenger (siderophore), to the MccE492m C-terminus. MceC and MceD derivatize enterobactin by C-glycosylation at the C5 position of a N-(2,3-dihydroxybenzoyl)serine (DHB-Ser) moiety and regiospecific hydrolysis of an ester linkage in the trilactone scaffold, respectively. Mcel and MceJ form a protein complex that attaches C-glycosylated enterobactins to the C-terminal serine residue of both a C₁₀ model peptide and full-length MccE492. In the enzymatic product, the C-terminal serine residue is covalently attached to the C4′ oxygen of the glucose moiety. Nonenzymatic and base-catalyzed migration of the peptide to the C6′ position affords the C6′ glycosyl ester linkage observed in the mature toxin, MccE492m, isolated from bacterial cultures.

Full text of DOI:10.1021/ja074650f

NIH Public Access
Author Manuscript
J Am Chem Soc. Author manuscript; available in PMC 2008 November 21.
Published in final edited form as:
J Am Chem Soc. 2007 November 21; 129(46): 14336–14347. doi:10.1021/ja074650f.
Biosynthetic Tailoring of Microcin E492m: Post-Translational
Modification Affords an Antibacterial Siderophore-Peptide
Conjugate
*
Elizabeth M. Nolan, Michael A. Fischbach, Alexander Koglin, and Christopher T. Walsh
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston,
MA 02115
Abstract
The present work reveals that four proteins, MceCDIJ, encoded by the MccE492 gene cluster are
responsible for the remarkable post-translational tailoring of Microcin E492 (MccE492), an 84-
residue protein toxin secreted by Klebsiella pneumonaie RYC492 that targets neighboring gram-
negative species. This modification results in attachment of a linearized and monoglycosylated
derivative of enterobactin, a nonribosomal peptide and iron scavenger (siderophore), to the
MccE492m C-terminus. MceC and MceD derivatize enterobactin by C-glycosylation at the C5
position of a N-(2,3-dihydroxybenzoyl) serine (DHB-Ser) moiety and regiospecific hydrolysis of an
ester linkage in the trilactone scaffold, respectively. MceI and MceJ form a protein complex that
attaches C-glycosylated enterobactins to the C-terminal serine residue of both aC model peptide
10
and full-length MccE492. In the enzymatic product, the terminal serine residue is covalently attached
to the C4′ oxygen of the glucose moiety. Non-enzymatic and base-catalyzed migration of the peptide
to the C6′ position affords the C6′ glycosyl ester linkage observed in the mature toxin, MccE492m,
isolated from bacterial cultures.
Introduction
Microcins are low molecular weight (< 10 kDa) ribosomal peptide toxins that enterobacteria
1
produce to inhibit the growth of competing bacteria and aid in host colonization. Many
microcin peptides exhibit unusual post-translational modifications that contribute to their
2
bactericidal effects. The microcin B17 (MccB17) precursor peptide undergoes tandem
heterocyclization, which generates thiazole and oxazole rings in the peptide backbone and
3,4
confers activity against DNA gyrase. Installation of adenosine-5′-monophosphate via a
phosphoramidate bond at the C-terminus of the microcin C7 (MccC7) precursor forms an
5
inhibitor of aspartyl-tRNA-synthethase. The post-translational modification of microcin
E492 (MccE492) creates a ribosomal peptide -nonribosomal peptide conjugate with a glucose
6
bridge. Together, these two scaffolds afford a toxin targeted to certain species of gram-
negative bacteria. The biosynthetic logic and remarkable post-translational maturation of
MccE492m (where m is for modified) are subjects of this work.
MccE492m, secreted by Klebsiella pneumonaie RYC492 and active against several species of
Enterobacteriaceae, is an 84-residue protein toxin exhibiting a striking C-terminal post-
translational modification comprised of a C-glycosylated and linearized enterobactin moiety
(Figure 1). Enterobactin (Ent, Figure 1), a cyclic trimer of N-2,3-dihydroxybenzoyl serine
(DHB-Ser), is a prototypic siderophore produced by gram-negative enterobacteria for iron
*Corresponding author: Christopher walsh@hms.harvard.edu.

Nolan et al.
Page 2
7
acquisition in the host environment. It is also the metabolic precursor to the recently
8,9
discovered salmochelins, C-glycosylated and linearized enterobactin congeners that may
10
confer more efficient iron acquisition in vivo because of their increased hydrophilicity and
11,12
ability to evade the mammalian protein lipocalin 2,
a component of the innate immune
13–15
response.
bacteria that express siderophore uptake pumps (FepA, Cir, Fiu) on the cell surface/outer
16,17
Post-translational modification of MccE492m enables it to target virulent
membrane,
making it a Trojan horse toxin. Following recognition, pore formation,
18–20
membrane depolarization, and cell death occur.
The mechanistic details of these
processes are unclear, but recent work indicates that the inner membrane protein components
21
of the mannose permease are required for MccE492m toxicity.
The MccE492 gene duster (Figure 2) encodes ten genes (mceABCDEFGJIH) that are necessary
22–25
and sufficient for MccE492m production in a strain that produces enterobactin.
mceA is
the structural gene for a 99- or 103-residue precursor protein (MceA) of ribosomal origin. The
precursor protein gets cleaved at amino acid 15 or 19, which yields the active 84-residue
23
MccE492 peptide. Microcin gene clusters often produce immunity proteins and the mceB
gene product is a 95-residue protein that confers resistance to MccE492(m) by an unknown
23
mechanism. Knock-out studies have implicated mceCDIJ in the maturation of MccE492m.
24
The mceG and mceH genes encode an ABC transporter and accessory protein, respectively,
that are necessary for microcin export. The functions of mceE and mceF are as yet undefined,
24
but the mceF gene product may also be involved in MccE492(m) export.
In this work, we examine several facets of MccE492m biosynthesis. We focus on four proteins,
MceCDIJ, and demonstrate that they catalyze the series of post-translational modifications
required for MccE492m formation. We were led to MceC and MceD because the MccE492
26–28
gene cluster shares some commonality with the iroA locus,
which encodes proteins
required for salmochelin synthesis (IroB, IroD, IroE) and transport (IroC, IroN). MceC and
29
30
MceD are IroB (C-glycosyItransferase) and IroD/IroE (esterase) homologs, respectively.
31
MceI is a homolog of HlyC, the acyltransferase required for hemolysin toxin activation, and
is essential for MccE492m formation. We therefore presumed that it is required for linking the
siderophore to the MccE492 C-terminus. Genetic studies have also shown that MceJ is
24,25
necessary for MccE492m maturation.
The function of MceJ was difficult to predict
because it has no known homologs other than its counterpart in the microcin H47 gene cluster
32
(MchC), but we speculated that it is also necessary for siderophore attachment. Herein we
report the over-production and purification of MceCDIJ, establish that MceC and MceD are a
C-glycosyltransferase and esterase, respectively, and show that MceIJ are sufficient to
derivatize both the C-terminal decapeptide of MccE492 and native MccE492 with
monoglycosylated enterobactins in vitro. We also demonstrate that the MceIJ product exhibits
the decapeptide attached to monoglycosylated enterobactin at the C4′ oxygen via an ester
linkage. This ester undergoes non-enzymatic and base-catalyzed rearrangement to the C6′
glycosyl ester, which is the observed connectivity in MccE492m isolated from bacterial
cultures.
Experimental
Materials and Methods
Oligonucleotide primers were synthesized by Integrated DNA Technologies (Coralville, IA.).
All chemicals were purchased from Sigma-Aldrich. Analytical and preparative HPLC were
performed on a Beckman System Gold (Beckman Coulter) instrument. A Vydac (Hesperia,
CA) small pore C18 (4.6 × 50 mm) column at 4 mL/min was generally employed for analytical
HPLC assays. A Vydac small pore C18 (4.6 × 250 mm) column at 1 mL/min was employed
for the MceIJ/lin-MGE assays and some MceIJ/MGE assays. A Vydac C18 (22 × 250 mm)
column at 10 mL/min was used for preparative HPLC. A Shimadzu LCMS-QP8000a outfitted
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Nolan et al.
Page 3
with a Higgins Analytical (Mountain View, CA) Sprite Targa C18 column (2.1 × 20 mm) at
0.5 mL/min was employed for LCMS. All calculated and found m/z ratios are the monoisotopic
values. Absorption at 220 and 316 nm was monitored for all liquid chromatography and 316
nm absorption traces are presented in all Figures unless otherwise noted. MALDI-TOF spectra
were collected on an Applied Biosystems Voyager mass spectrometer. With the exception of
the NOESY spectra that were acquired as described below in the “Structural Elucidation of
MceIJ Products” section, NMR spectra were collected on a Varian 600 MHz instrument and
referenced to internal solvent peaks. A Cary 50 Bio scanning spectrometer was used to record
absorption spectra. The concentrations of the siderophore stock solutions were verified using
33
−1
−1
the reported extinction coefficients for enterobactin (316 nm, 9500 M cm ), linear
−1
−1
3
−1
−1
enterobactin (315 nm, 9700 M cm ) and [Fe(enterobactin)] ” (338 nm, 15100 M cm )
with the assumption that the glucose moieties would have no effect on siderophore absorption.
Siderophore stock solutions were generally prepared in DMSO (~3 to ~10 mM), divided into
aliquots and stored at −20 °C. The Bradford assay was employed to determine protein
34
concentration. The decapeptide, SATSSSGSGS, was prepared by standard Fmoc solid phase
peptide synthesis by staff at the Biopolymers Facility at Harvard Medical School. Its purity
and identity were verified by analytical HPLC and LCMS. Concentrations of peptide stock
solutions were verified by quantitative amino acid analysis (Dana Farber Cancer Institute).
Details for substrate preparations are included in the Supporting Information section.
Cloning, Overexpression and Purification of MceD
The mceD gene was PCR amplified from pJEMIS using the forward primer 5′-
ggaattccatatgccatttatgaggaatcatccatc-3′ (NdeI restriction site underlined) and reverse primers
5′-gatcctcgagttacacatctgataatccatcgataattg-3′ (pET-28b, XhoI restriction site underlined) and
5′-gatcctcgagcacatctgataatccatcgataattg-3′ (pET-29a, XhoI restriction site underlined). PCR
reactions were performed with Pfu Turbo DNA polymerase (Stratagene) and the amplified
gene sequences were digested with NdeI and XhoI (New England Biolabs), ligated into
pET-28b and pET-29a expression vectors using T4 DNA ligase (New England Biolabs) and
transformed into E. coli TOP10 cells (Stratagene). The identities of the resulting pET-28b (N-
His) and pET-29a (C-His) constructs were confirmed by DNA sequencing. A discrepancy (G
to A) at position 5378 with the published sequence was observed for the pET28b-mceD
construct. Expression constructs were transformed into E. coli BL21(DE3) cells, grown to
saturation in LB medium supplemented with 50 μg/mL kanamycin at 37 °C and diluted 1:100
into LB medium containing 30 μg/mL kanamycin. The cultures were incubated at 37 °C with
shaking, induced with 400 μM IPTG at OD = 0.5–0.6, and then incubated at 15 °C for ~20
600
h. Cells from 4 L of culture were pelleted by centrifugation (6,000 rpm × 10 min), resuspended
in 60 mL of buffer A (20 mM Tris-HCl pH 8, 500 mM NaCl and 10 mM MgCl ) and
2
homogenized. The homogenate was passed through a cell disrupter (Avestin EmulsiFlex-C5)
twice at 5,000 – 10,000 psi and the lysate was clarified by ultracentrifugation (35,000 rpm ×
35 min). The supernatant was incubated with ~1.5 mL of Ni-NTA resin (Qiagen) for 2 h at 4
°C and the mixture was centrifuged (3,000 rpm × δ min). The unbound fraction was discarded
and the Ni-NTA resin resuspended in 10 mL of buffer B (20 mM Tris-HCl pH 8, 500 mM
NaCl and 10 mM MgCl , δ mM imidazole) and loaded onto a column and washed with 10 mL
2
buffer B. MceD was eluted from the column using a stepwise imidazole gradient (25 to 200
mM). SDS-Page analysis (4–15% Tris-HCl gel, BioRad) was employed to ascertain the
presence and purity of MceD in each fraction. Fractions containing pure MceD were combined
and dialyzed twice against buffer C (25 mM Tris-HCl pH 8, 50 mM NaCl, 1 mM DTT, 10%
glycerol). This procedure afforded yields of >15 mg/L for both N- and C-terminally tagged
MceD. Gel filtration (Supradex 200) indicated that the protein is monomeric. The protein was
frozen in liquid N and stored at −80 °C. Initial activity assays indicated that both N-terminally
2
and C-terminally His tagged MceD were active. All subsequent assays were conducted with
6
the N-terminally tagged enzyme.
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Cloning, Overexpression and Purification of MceC
The mceC gene was subcloned from pJEM15 using the forward primer 5′-
ggaattccatatgcgtattctctttattggccctcc-3′ (NdeI) and reverse primer 5′-
gatcctcgagttgccagatggttttcagtttcgc-3′ (XhoI). Pfu Turbo DNA polymerase was employed for
PCR reactions and the amplified genes were digested with NdeI and XhoI, ligated into the
pET-29a expression vector using T4 DNA ligase and transformed into E. coli TOP10 cells.
The identity of the construct was confirmed by DNA sequencing. E. coli BL21(DE3) cells
were transformed with the construct and grown to saturation in LB media containing 50 μg/
mL kanamycin. MceC was subsequently overexpressed, purified and stored as described for
MceD with one modification. During initial purifications, MceC precipitated within minutes
of elution from the Ni-NTA column. Immediate addition of enterobactin to fractions containing
MceC remedied this problem. For a typical Ent addition, a 700-μL aliquot of Ent (10 mM in
DMSO) was added to 12 mL of eluted MceC. This mixture was immediately transferred to a
dialysis cassette (10 kDa cut-off) and dialyzed twice against buffer C. This approach afforded
MceC in yields of ~4 mg/L.
Cloning, Expression and Purification of MceIJ
The fragment encoding mceIJ was subcloned from pJEM15 as a bicistronic operon using the
forward primer 5′-ggaattccatatggtaacggagaaatacagtcaaatg-3′ (NdeI) and the reverse primers
5′-gatcctcgagtcaaagttctttctgtgtctccggg-3′ (XhoI, pET-28b) and 5′-
gatcctcgagaagttctttctgtgtctccggggcc-3′ (XhoI, pET-22b). The genes were amplified and ligated
into E. coli expression vectors as described for MceD above. The pET-28b-mceIJ vector
encodes MceJ as a N-terminal His fusion and untagged MceI. The pET-22-mceIJ vector
6
encodes MceI as a C-terminal His fusion and untagged MceJ. The proteins were over-
6
expressed and purified as described for MceD. SDS-Page analysis indicated that both MceI
and MceJ co-elute from the Ni-NTA column regardless of which component bears a His tag.
6
All subsequent work was conducted with the MceIJ complex in which the His tag is fused to
6
MceJ, which was generally obtained in yields of >3 mg/L. Gel filtration analysis (Supradex
200) of MceIJ showed a retention volume of ~150 mL, which is consistent with a molecular
weight range of 150–200 kDa and suggests a tetrameric A B state. Several variations in the
2
2
MceIJ purification protocol were tested and had negligible effect on activity. In particular, the
presence of DTT has no effect on MceIJ activity.
Large-Scale Enzymatic Preparation of MceC Glycosylation Products
A 48 mL aqueous solution containing 75 mM Tris-HCl pH 8, δ mM MgCl 2.5 mM tris(2-
2
carboxyethyl)phosphine hydrochloride (TCEP), 100 μM enterobactin, and 600 μM uridine
diphosphoglucose (UDP-Glc) was prepared and divided into 48 aliquots of 900 μL. To each
aliquot was added 100 μL of 10 μM MceC in 75 mM Tris-HCl pH 8 buffer. The solutions were
mixed with a pipette, incubated at room temperature for 90 min, and each quenched with 500
μL of 2.5 N HCl in MeOH. The reactions were centrifuged (13,000 rpm × 10 min), combined
and concentrated to ~16 mL on a lyophilizer. The concentrated material was centrifuged
(13,000 rpm × 10 min) again. MGE (1.3 mg) and DGE (0.9 mg) were obtained as white powders
following preparative HPLC using a solvent gradient of 0 to 40% B over 40 min (solvent A,
1
0.2% TFA/H O; solvent B, 0.2% TFA/MeCN) and lyophilization. H NMR (CD OD, 600
2
3
MHz) δ for MGE: 7.38 (1H, s), 7.16 (2H, dd), 7.00 (1H, d), 6.94 (2H, dq), 6.72 (2H, td), 5.03
(3H, m), 4.67 (3H, m), 4.59 (3H, m), 4.01 (1H, d), 3.86 (1H, d), 3.69 (1H, m), 3.42 (2H, m),
3.36 (2H, m); δ for DGE: 7.39 (1H, dd), 7.27 (1H, dd), 7.06 (2H, s), 6.96 (1H, dd), 6.74 (1H,
t), 5.05 (3H, m), 4.70 (3H, m), 4.59 (3H, m), 4.02 (2H, m), 3.86 (2H, dd), 3.71 (2H, m), 3.27–
+
3.47 (8H, m). MS (ESI): [M+H] m/z calc 832.2 (MGE), 994.3 (DGE); found, 832.1 (MGE),
994.3 (DGE).
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Large-Scale Enzymatic Preparation of MceD Hydrolysis Products
For MGE: A portion (15.6 mg, 18.6 μmol) of MGE was dissolved in 200 μL of DMSO and
diluted to a final volume of 15 mL with 75 mM Hepes pH 7.5. A 135 μL aliquot of MceD (71
μM in buffer C) was added and the reaction was mixed thoroughly and incubated for 20 min
at room temperature. The reaction was quenched with addition of 2.5 N HCl in MeOH (7 mL),
vortexed and filtered through a 0.2 μm membrane. The products were separated and purified
by preparative HPLC using a solvent gradient of 0 to 40% B in 40 min (solvent A, 0.2% TFA/
H O; solvent B, MeCN), which afforded linear MGE (lin-MGE, 2.5 mg) and the glycosylated
2
DHB-Ser dimer (Glc-Dimer, 1.8 mg) as white powders following lyophilization. Product purity
1
was verified by analytical HPLC. H NMR (CD OD, 600 MHz) δ for lin-MGE: 7.45 (1H, d),
3
7.27 (2H, qd), 7.04 (1H, d), 6.93 (2H, dq), 6.70 (2H, td), 5.05 (1H, q), 5.00 (1H, q), 4.81 (1H,
d), 4.76–4.81 (2H, m), 4.69 (1H, t), 4.60 (1H, m), 4.51 (1H, m), 4.05 (1H, d), 3.96 (1H, m),
3.87 (2H, m), 3.71 (1H, m), 3.44 (2H, m), 3.39 (2H, m); for Glc-Dimer: 7.44 (1H, d), 7.32 (1H,
dd), 7.04 (1H, d), 6.94 (1H, dd), 6.73 (1H, t), 5.04 (1H, m), 4.89 (2H, m), 4.78 (1H, t), 4.52
(1H, m), 4.04 (2H, m), 3.95 (1H, d), 3.93 (1H, d), 3.87 (1H, d), 3.85 (1H, d), 3.72 (2H, m),
+
3.46 (2H, m), 3.36 (2H, m). MS (ESI) [M+H] m/z calc 850.2 (lin-MGE), 627.2 (Glc-dimer);
found, 850.2, 627.4. For DGE: As described for MGE except that DMSO was not employed
because DGE (16 mg, 16 μmol) exhibited sufficient water solubility. Linear DGE (lin-DGE)
and the glycosylated DHB-Ser dimer were obtained as white powders in 6.5 and 4.0 mg
1
quantities, respectively. H NMR (CD OD, 600 MHz) δ lin-DGE: 7.45 (1H, s), 7.40 (1H, s),
3
7.30 (1H, d), 7.03 (2H, m), 6.94 (1H, d), 6.72 (1H, t), 5.08 (1H, m), 5.00 (1H, t), 4.68 (1H, t),
4.56 (1H, m), 4.51 (1H, m), 4.04 (2H, t), 3.96 (1H, m), 3.86 (3H, m), 3.70 (2H, m), 3.35–3.49
+
(8H, m). MS (ESI) [M+H] m/z calc 1012.3 (lin-DGE), 628.2 (Glc-Dimer); found, 1012.2,
627.3.
Large-Scale Preparation of MceIJ Reaction Products
A 102 mL solution containing 75 mM Tris-HCl pH 8, 5 mM MgCl , 2.5 mM TCEP, 5 mM
2
100 μM MGE, 550 μM C (C-terminal decapeptide, SATSSSGSGS) and was divided into
10
202 aliquots of 460 μL. A 40 μL portion of ~20 μM MceIJ in buffer C was added to each aliquot
and the reactions were incubated at room temperature for 7.25 h. Each reaction was quenched
with 100 μL of 0.3% TFA/H O and centrifuged (13,000 rpm × 10 min). The supernatants were
2
combined and concentrated on a lyophilizer. The two products from the MceIJ-catalyzed
reactions were separated by preparative HPLC using a solvent gradient of 0 to 40% B over 40
min (solvent A, 0.2% TFA/H O; solvent B, 0.2% TFA/MeCN). The collected fractions were
2
lyophilized to dryness, which afforded white powders (1.9 mg, product 1; 3.2 mg, product 2).
The purity of each product was verified by analytical HPLC. 2-dimensional (2-D) NMR was
+
employed to determine product identity as described below. MS (ESI) [M+H] ra/z calc,
1640.5; found, 1640.2 for both products.
Structural Elucidation of MceIJ Products
The products from the large-scale MceIJ reaction were structurally characterized using 2-D
NMR spectroscopy. Data acquisition was performed on a Bruker Avance 500 spectrometer
1
with a H-Lamor frequency of 500.13 MHz equipped with a triple resonance z-gradient
cryogenic probe. The sample temperature was 298 K. The samples were dissolved in 500 μL
of 9:1 H O/D O (pH ~ 4) for final concentrations of 1.9 (peak 1) and 3.8 mM (peak 2). For
2
2
1
spectral analysis and resonance assignment, a sect of two 2D- H homonuclear NOESY spectra
with correlation via dipolar coupling was recorded for each compound. A water gate W5 pulse
sequence was applied for water suppression. For each set of experiments, the mixing time
35
was varied from 80 to 250 ms to distinguish between short- and long-range interactions and to
support the resonance-specific assignments. All spectra were processed with XWINNMR 3.1
36
and analyzed with SPARKY 3.111. For spectra processing, a square sine bell apodization
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Nolan et al.
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function was used for Fourier Transformation. For both compounds, all proton resonances were
assigned with the exceptions of the hydroxyl protons from the hexose, the serine sidechains
and the benzoic acid residues of the 2,3-DHB units, which could not be observed in aqueous
solution. Starting with the unambiguous low field amide proton resonances from the serine
residues of the three DHB-Ser units, the aromatic moieties of the DHB units were assigned by
intra- and inter-residual NOEs. NOE correlations of one DHB unit into the hexose residue and
further to the attached peptide supported the sequential assignment of the attached peptide. The
peptide was completely sequentially assigned using the amide-to-amide proton correlations.
Initial Activity Assays with MceC
Initial activity assays with MceC were conducted in buffer D (75 mM Tris-HCl pH 8, δ mM
MgCl and 2.5 mM TCEP). For a typical assay, a 500 μL solution containing 100 μM
2
siderophore, 600 μM UDP-Glc and 1 μM MceC was prepared, mixed and incubated at room
temperature. A solution of 2.5 N HCl in MeOH (75 μL) was used to quench reaction aliquots
(75 μL) at various time points. The quenched solutions were centrifuged (13,000 rpm × 10
min) and analyzed by HPLC. A solvent gradient of 0 to 40% B in 8 min (solvent A, 0.1% TFA/
H O; solvent B, MeCN) was employed. Product identity was confirmed by co-elution with
2
hydrolysis products of IroB-catalyzed reactions, LCMS and NMR.
Kinetic Investigations of MceC
Kinetic investigations of MceC-catalyzed glycosylation of Ent, linear Ent (lin-Ent) and MGE
were conducted in buffer D. To determine k and K for the donor substrate, the UDP-Glc
cat
M
concentration was varied (0 – 256 μM) in the presence of 100 μM Ent and 200 nM MceC, and
the reactions (100 μL volume) were quenched at t = 20 min with 75 μL of 2.5 N HCl in MeOH.
To determine the kinetic parameters for the acceptor substrates, the UDP-Glc concentration
was held constant at 500 μM and the siderophore concentrations were generally varied from
0 – 256 μM with a MceC concentration of 100 (Ent) or 200 (MGE, lin-Ent) nM. The 100-μL
reactions were quenched at t = 1 (Ent), 7 (MGE) or 8 (lin-Ent) min with 75 μL of 2.5 N HCl
in MeOH. The quenched solutions were centrifuged (13,000 rpm × 10 min), stored on ice and
analyzed by HPLC using a solvent gradient of 0 to 40% B in 8 min (solvent A, 0.1% TFA/
H O; solvent B, MeCN). Product quantification was based on the area of the 316 nm absorption
2
peaks. All kinetic runs were repeated in triplicate and the data fit to the Michaelis-Mention
equation. The Ent present as a result of the MceC preparation was taken into account in total
[Ent] used in the analysis of MceC glycoslyation of Ent.
Kinetic Investigations of IroB
Kinetic studies of IroB-catalyzed glycosylation of Ent, lin-Ent, MGE, lin-MGE and DGE were
conducted as described for MceC. The UDP-Glc concentration was 500 μM and the
siderophore concentrations were generally varied from 0 – 256 μM in the presence of 100 nM
(Ent, MGE, DGE), 200 (lin-Ent) nM or 1 μM (lin-MGE) IroB. The 100 μL reactions were
quenched at t = 2.5 (Ent), 4 (MGE), 20 (lin-Ent, lin-MGE) or 30 (DGE) min with 75 μL of 2.5
N HCl in MeOH and analyzed as described for MceC.
Initial Activity Assays with MceD
Initial activity assays with MceD were conducted in 75 mM Hepes buffer adjusted to pH 7.5.
In general, a 500 μL solution containing 32 μM siderophore and 20 nM MceD was employed.
Reaction aliquots (100 μL) were quenched at t = 0, 2, 8, 25 min with 2.5 N HCl in MeOH (100
μL), centrifuged (13,000 rpm × 10 min) and stored on ice. In all cases, the reaction progress
was monitored by analytical HPLC using a solvent gradient of 0 to 40% B in 8 min (solvent
A, 0.1% TFA/H O; solvent B, MeCN). Product identity was confirmed by co-elution with
2
hydrolysis products of IroD catalyzed reactions and by LCMS.
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Kinetic Investigations of MceD
Kinetic studies of MceD-catalyzed hydrolysis of apo Ent, MGE, DGE, lin-MGE and lin-DGE
3−
3−
3−
and [Fe(Ent)] , [Fe(MGE)] and [Fe(DGE)] were conducted at room temperature in 75
mM Hepes buffer adjusted to pH 7.5. The enzyme concentration was 5 nM and the substrate
concentration was generally varied from 0 – 256 μM for apo and from 0 – 32 μM for ferric
substrates. Each 100-μL reaction was quenched with 50 μL of 2.5 N HCl in MeOH at t = 30
sec, centrifuged (13,000 rpm × 10 min) stored on ice and analyzed as described above. Negative
control reactions with no enzyme were conducted to determine if background hydrolysis
30
needed to be taken into account. This treatment was necessary for Ent and lin-Ent. Kinetic
studies of the MceD-catalyzed hydrolysis of C -C6′-MGE and C -C4′-MGE were conducted
10
10
in a similar manner except that the reactions were quenched at t = 1 min. Some (<10%)
migration of the C peptide from the C4′ to the C6′ position was observed when C -C4′-
10
10
MGE was employed as a substrate and the area of both peaks were considered in the data
analysis.
Activity Assays with McelJ
Activity assays with MceIJ were generally conducted at pH 8 (75 mM Tris-HCl) and in the
presence of 2.5 mM TCEP and 5 mM ATP with 100 μM MGE/lin-MGE, 550 μM C peptide
10
and 2 μM MceIJ. The reactions (300 μL) were incubated at room temperature, quenched at
various time points with an equal volume of 0.6% TFA/H O, vortexed and centrifuged (13,000
2
rpm × 10 min). The reaction progress was monitored by analytical HPLC using a solvent
gradient of 0 to 40% B (MGE) or 15 to 35% B (lin-MGE) where solvent A is 0.2% TFA/H O
2
and solvent B is 0.2% TFA/MeCN. Product identities were determined by LCMS and NMR
as described above.
Results and Discussion
MccE492m exhibits a number of remarkable structural features (Figure 1). The antibiotic
illustrates an intersection of ribosomal and non-ribosomal peptide synthesis. It bears an unusual
post-translational modification at the C-terminus comprised of an ester linkage between the
ribosomally-derived MccE492 peptide and the C6′ oxygen of the non-ribosomally synthesized
monoglycosylated and linearized enterobactin (lin-MGE). The glucose moiety in MccE492m
is a bridging element that connects its two functional units, the toxic microcin peptide and the
siderophore recognition element. The serine-rich C-terminus of MccE492 is another striking
feature shared by several other microcins including MccH47, MccI47 and MccM (Figure 2).
37–42
This commonality may point to similar post-translational modifications at the C-termini
and a general strategy for destroying competing bacterial species that express siderophore
43,42
receptors.
The biosynthesis of the siderophore moiety itself requires multiple layers of
secondary metabolism. Its central scaffold, enterobactin, is the product of the enterobactin
synthetase and requires both C-glycosylation and linearization for MccE492m maturation.
Unveiling the biosynthetic tailoring of MccE492m provides a foundation for future studies of
structurally related microcins, including MccH47, MccI47 and MccM, and a platform for the
design of new Trojan horse antibiotics.
Expression and Purification of MceC, MceD and MceIJ
We chose first to investigate MceC and MceD based on their respective amino acid sequence
homology to IroB (C-glycosyItransferase) and IroD/IroE (esterases) from the iroA gene cluster.
IroB carries out regiospecific C-glycosylation of enterobactin at the C5 positions of the DHB
rings and IroD/E catalyze hydrolysis of the ester linkages in the enterobactin macrolactone to
29,30
afford linearized congeners.
IroD and IroE both accept glycosylated enterobactins as
substrates and IroD processes these forms regioselectively.
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Nolan et al.
Page 8
The mceC and mceD genes were subcloned from pJEM15 into E. coli expression vectors as
either N- and/or C-terminal His fusions. MceD (45.8 kDa, 414 aa) was over-expressed in E.
6
coli BL21(DE3) cells as N- and C-terminally His fusions and purified by Ni-NTA affinity
6
chromatography with yields of >15 mg/L (Figure 3). MceC (39.9 kDa, 370 aa) was over-
expressed as a C-terminal His fusion and purified in a similar manner (Figure 3), but exhibited
6
very poor solubility following elution from the Ni-NTA column. We reasoned that addition of
enterobactin, a putative substrate of MceC, might stabilize the enzyme and afford solubility.
We subsequently added Ent to several MceC-containing fractions and immediately dialyzed,
which conferred solubility.
24,25
Genetic studies revealed that mcel and mcej are required for MccE492m production.
In
related work, initial attempts to overexpress and purify MceIJ homologs from the microcin
H47 gene cluster, MchD and MchC, did not yield appreciable quantities of soluble protein.
43
Co-expression of MchC and MchD afforded both proteins as a soluble complex. We
therefore subcloned the bicistronic operon encoding mceIJ with a His tag fused either to the
6
C-terminus of MceI (19.8 kDa, 163 aa) or the N-terminus of MceJ (59.1 kDa, 524 aa). The
proteins were over-expressed in E. coli BL21(DE3) cells and purified using Ni-NTA column
chromatography. SDS-PAGE analysis showed that MceI-C-His pulled down MceJ and that
6
MceJ-N-His pulled down Mcel, indicating formation of a Mcelj complex (Figure 3). Gel
6
filtration supported complex formation and the retention volume suggested an A B tetramer,
2
2
although further work is required to verify this notion.
MceC is a C-Glycosyltransferase that Modifies Apo Siderophores
29
Based on the MccE492m structure and our previous studies of homolog IroB, we anticipated
that MceC would catalyze C-glycosidic bond formation between C1′ of the glucose and C5 of
the DHB ring to form monoglycosylated enterobactin (MGE). Because the Ent derivative in
MccE492m has only one glucose moiety, we hypothesized that MceC might differ from IroB
in its ability to successively glycosylate the Ent scaffold. Incubation of IroB with Ent and UDP-
Glc resulted in the distributive formation of three products, mono-, di- and triglycosylated
enterobactin. MceC, in contrast, might prefer to glycosylate Ent only once, generating a product
that would be on-pathway to MccE492m.
Preliminary activity assays revealed that incubation of MceC with Ent and UDP-Glc for t < 30
min resulted in formation of a new peak in the analytical HPLC trace with a retention time and
+
mass consistent with MGE ([M+H] ra/z calc, 832.2; found, 832.1) (Figure 4). Following
accumulation of MGE, a second peak appeared at t > 30 min with a retention time and mass
+
consistent with DGE ([M+H] m/z calc, 994.3; found, 994.3). MceC did not generate TGE
(Figure SI, Supporting Information), a novel enterobactin derivative isolated from IroB-
29
1
catalyzed reactions in vitro, even in the presence of 1 mM UDP-Glc (t = 180 min). H (MGE,
DGE) and HMBC NMR spectra (DGE) analysis of the products obtained from a large-scale
MceC-catalyzed glycosylation of Ent confirmed the C-glycosidic linkage between the C1′ of
the glucose moiety and C5 of the DHB unit (Supporting Information). The spectroscopic data
29
for DGE are in agreement with those of DGE isolated from an IroB-catalyzed reaction and
the structure of salmochelin S4, which was recently isolated from Salmonella enterica.
9
Because the enterobactin scaffold in MccE492m is linearized, we investigated whether MceC
would also accept linearized enterobactin (lin-Ent) as a substrate. Incubation of MceC with
lin-Ent and UDP-Glc resulted in the formation of a new peak in the analytical HPLC trace with
+
a mass consistent with linear MGE (calc [M+H] m/z calc, 850.2; found, 850.5) and retention
time equivalent to that of lin-MGE isolated from MceD-catalyzed hydrolysis of MGE (vide
infra). We also surveyed lin-MGE and linearized DGE (lin-DGE), prepared by large-scale
MceD-catalyzed reactions, and found that these siderophores are not good substrates for MceC.
No glycosylation of lin-MGE or lin-DGE was observed.
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Nolan et al.
Page 9
Enterobactin is an iron-chelator produced by bacteria existing in iron-limiting conditions and
could therefore be a substrate for MceC in its apo or holo form. We therefore questioned if
3−
3−
3−
MceC would also recognize [Fe(Ent)] , [Fe(MGE)] , and [Fe(DGE)] . Initial activity
assays indicated that the ferric siderophores are not viable substrates for MceC. Incubation of
MceC with a ferric siderophore and UDP-Glc resulted in either negligible or no glycosylation
of the DHB moieties (t = 60 min). This behavior is analogous to that of IroB, which does not
3−
44
accept [Fe(Ent)] or its glycosylated congeners.
To gain further insight into the substrate specificity of MceC and its homolog IroB, kinetic
studies were undertaken with the apo siderophores and the resulting data are listed in Table 1
(Figures 4 and S1). The kinetic parameters for the acceptor substrates reveal that the catalytic
efficiency of MceC decreases with either glycosylation or linearization of the enterobactin
scaffold with Ent > MGE > lin-Ent. A comparison of data for enterobactin and MGE indicates
a ~40-fold reduction in k /K for the second glycosylation event. Likewise, linearization of
cat
m
the Ent scaffold results in an ~4.4-fold decrease in k and ~19-fold increase in K , affording
cat
m
an overall ~97-fold reduction in k /K for lin-Ent relative to enterobactin. Substrate inhibition
cat
m
was also observed with [lin-Ent] > 64 μM. In total, these data indicate that enterobactin is the
preferred substrate for MceC and that MceC acts prior to MceD in MccE492m maturation.
The kinetic data for IroB show generally similar trends. IroB has a broader substrate scope
than MceC and accepts DGE and lin-MGE, substrates with greater steric bulk. The catalytic
efficiency decreases with glycosylation and linearization in the order Ent > MGE > DGE >
lin-Ent > lin-MGE (Table 1, Figure S1).
MceD is an Esterase and Hydrolyzes Apo and Ferric Siderophores
MceD is a homolog of IroD/IroE, the enterobactin hydrolases encoded by the iroA gene cluster.
Although IroD and IroE both accept apo and ferric enterobactin and their glycosylated
3−
3−
congeners, IroD prefers to process [Fe(MGE)] /[Fe(DGE)] whereas IroE preferentially
hydrolyzes the apo forms. IroD cuts MGE and DGE regioselectively and readily degrades the
linear DHB-Ser trimer products to monomers and dimers. IroE, in contrast, hydrolyzes
30
glycosylated Ent species with no regioselectivity and cuts only once to yield trimers. With
the MccE492m structure in mind, we anticipated that MceD would exhibit the regioselectivity
of IroD and hydrolyze the macrolactone ring once in an IroE-like manner.
Incubation of Ent with MceD in pH 7.5 buffer resulted in hydrolysis of the lactone scaffold
(Figure 5). At t > 2 min, several Ent hydrolysis products were observed in the analytical HPLC
trace, which correspond to lin-Ent and its dimeric and monomeric derivatives, arising from
two and three hydrolytic cuts, by retention time and MS analysis (Table S1). This distribution
indicates that the initial lin-Ent product, generated by Ent hydrolysis, is also a good substrate
for MceD. This behavior is similar to that of IroD, which readily hydrolyzes the ester linkages
30
in both cyclic and linear Ent trimers.
Initial assays with MGE and DGE under the same conditions indicated that the presence of
glucose moieties alters the product distribution of MceD (Figures 5, S2). MGE and DGE are
both hydrolyzed by MceD, but much more readily than lin-MGE and lin-DGE. As a result,
build-up of lin-MGE and lin-DGE occurs followed by some formation of the dimeric and
monomeric hydrolysis products. The kinetic data corroborate these observations (Table 2).
Comparisons of k /K for the apo substrates reveal that (i) MceD hydrolyzes Ent and lin-Ent
cat
m
with comparable efficiency, (ii) successive glycosylation of Ent results in increases in both
and K , yielding an overall >2-fold decrease in catalytic efficiency and (iii) linearization
k
cat
m
of the MGE scaffold results in a ~50-fold decrease in MceD efficiency relative to the
macrolactone because of a ~ 19-fold decrease in k and ~3-fold increase in K . The ~50-fold
cat
m
preference of MceD for MGE over lin-MGE suggests that MceD is capable of producing a
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Nolan et al.
Page 10
13
1
13
linearized trimer like the one in MccE492m. NMR spectra ( H and C 1-D, HMBC, C-
HSQC) of the trimeric and dimeric products from MGE and DGE hydrolysis are in agreement
30
with those obtained for the analogous species obtained from IroD-catalyzed reactions, which
indicates that both enzymes cleave MGE and DGE with the same regioselectivity (Figure 5).
6
This mode of cutting affords the lin-MGE isomer observed in MccE492m.
MceD also hydrolyzes the ferric siderophores with product distributions that mirror those
observed for the apo congeners (Figure S3). Prior studies of the related esterase, Fes, came to
30,
contradictory conclusions depending on assay conditions and subsequent interpretation.
45,46
Because siderophores are quickly exported for iron acquisition following biosynthesis,
the intracellular concentrations are most likely lower than theK value. As a result, the apparent
m
second-order rate constant, k /K , should be considered when quantifying the throughput of
cat
m
siderophore-tailoring enzymes. The relative catalytic efficiencies for apo and ferric Ent, MGE
and DGE reveal that MceD prefers the apo congeners (Table 2). Although the K values are
m
generally lower for the ferric siderophores than their apo counterparts, the k values are
cat
substantially reduced and the net effect is a ≤5-fold decrease in k /K . A similar trend was
cat
m
30
previously observed for IroE.
MceIJ is Responsible for Post-Translational Modification of MccE492
The post-translational modification that gives rise to MccE492m is unusual because it involves
modification of the peptide C-terminus. Other C-terminal modifications have been documented
47
48–
in the esterification of small GTPases, including Ras, and in the structure of MccC7.
50
Some microcins, including MccH47, MccI47 and MccM, exhibit serine-rich C-terminal
regions like that of MccE492 (Figure 2) and may exhibit post-translational modifications
identical to that of MccE492m. A recent study of microcin MccH47/ColV chimeras points
42
to a modular structural organization where N- (toxin) and C-terminal (recognition) domains
51
can be exchanged readily to yield active bacteriocins. This modularity may have played a
role in the evolution of this family of peptide toxins.
Previous genetic studies suggest that MceI and MceJ catalyze the post-translational
24
modification that converts MccE492 to MccE492m. Removal of mceI from the MccE492
gene cluster prevents maturation of MccE492. Its protein product, MceI, is a 19.8 kDa enzyme
with homology to HlyC, the acyltransferase responsible N-acylation of lysine residues in
31
hemolysin toxin, and MchD, which is required for MccH47 maturation. The MccE492 gene
cluster also encodes MceJ, a 59.1 kDa protein of unknown function and with no known
homologs other than its counterpart in the microcin H47 gene cluster (MchC), which has also
been implicated in MccE492m maturation.
We therefore subcloned the bicistronic operon encoding MceIJ and found that the two proteins
form a complex. We also designed a model system for assaying MceIJ activity. We
hypothesized that the serine-rich portion of the MccE492 C-terminus might be sufficient for
recognition by MceIJ, and utilized a model decapeptide comprised of the last ten residues of
MccE492, SATSSSGSGS (C ), as a peptide substrate analog. We employed MGE rather than
10
lin-MGE as the siderophore substrate because (i) the preparation of MGE requires one less
enzymatic reaction, (ii) MGE decomposes less readily than lin-MGE in buffered aqueous
solution at room temperature, and (iii) we were uncertain as to the order of MceD and MceIJ
action during MccE492m biosynthesis.
Initial activity assays with this model system revealed several important facets of MccE492m
assembly: (i) MceIJ recognizes the C peptide substrate, (ii) MceIJ accepts MGE as a
10
substrate, (iii) only ATP and MgCl are required as co-factors for attachment of MGE to the
2
decapeptide, and (iv) two new peaks, 1 and 2, form in the analytical HPLC trace following
incubation of MceIJ with C , MGE, ATP and MgCl (Figure 6). Isolation and MS analysis
10
2
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Nolan et al.
Page 11
of the two product peaks indicated that they have the same molecular weight equivalent to
C +MGE-H O that is consistent with attachment of MGE to the C peptide through an ester
10
2
10
+
linkage ([M+H] m/z calc, 1640.5; found, 1640.2 (1), 1640.2 (2)).
To gain further insight into the origins of peaks 1 and 2, both products were obtained in multi-
milligram quantities and in high purity from large-scale MceIJ-catalyzed reactions (Figure 7).
A series of activity assays indicated that (i) formation of 1 is enzyme-dependent, (ii) formation
of 2 from 1 does not require enzyme, (iii) conversion of 1 to 2 does not happen in water (pH
~ 5), but occurs in solutions buffered in the pH 7 to 9 range, (iv) the conversion rate of 1 to 2
is accelerated by base, and (v) incubation of peak 2 in the absence or presence of MceIJ does
not result in significant formation of 1 in the pH range of 5.5 to 9 (Figure S4). In particular,
analytical HPLC studies showed that a 50 μM solution of 1 is converted to ~20% (pH 7), ~50%
(pH 8) or ~80% (pH 9) 2 following a 30 min incubation at room temperature. Incubation of
2 at room temperature for 30 min resulted in ~10% conversion to 1 in the pH 7 to 9 range.
To verify that MceIJ is indeed responsible for the MccE492m post-translational modification,
an assay with MceIJ, ATP, MgCl , MGE and native MccE492, isolated from E. coli VCS257
2
6
harboring pJAM434 according to a published protocol, was performed. A new peak in the
HPLC trace exhibiting catecholate (316 nm) absorption was observed and MALDI-TOF
+
analysis indicated a mass consistent with MccE492+MGE-H O ([M+H] m/z calc, 8700.2;
2
+
found, 8704.4; [M+H] for unmodified MccE492 m/z calc, 7887.5; found, 7892.3).
Structural Analysis of the MceIJ Reaction Products: Initial Reaction at the C4′ Hydroxyl of
MGE
We reasoned that peaks 1 and 2 are two isomers of the C -MGE structure with one exhibiting
10
C
linked to MGE through the C6′ oxygen on the glucose moiety as seen in the MccE492m
10
1
structure and the other a novel isomer. We employed 2-D- H-homonuclear NMR to determine
the product identities. Table S2 lists the resonance assignments for all observed protons in 1
and 2 and sections of the corresponding NOESY spectra are presented in Figure 7. The
6
published assignment of MccE492m(75–84) was largely reproduced for both compounds.
The C peptide was assigned using amide-to-amide proton correlation and the sequence was
10
corroborated by identification of the distinct side chain proton resonances of the Ala-2 2 Hβ
methyl protons and the Thr-3 Hγ proton. The structures of the MGE moieties in 1 and 2 were
confirmed as enterobactin linked to a hexose by a C-glycosidic bond between the C1′ atom of
the sugar and the C5 position of a DHB-Ser unit. The C-C bond between C5 of DHBS1 and
C1′ of the glucose was confirmed by the observed NOE correlations between the H4 and H6
protons of DHBS1 and the H1′ proton of the sugar. The absence of the H5 proton resonance
of DHBS1 and the identifiable H6-H5 and H4-H5 correlations for DHBS2 and DHBS3 further
verifies the nature of the C-glycosidic bond.
The proton resonances and NOE correlations for the sugar moieties in 1 and 2 reveal key
differences. For 2, correlations between H1′-H3′, H3-′H5′ and H5′-H1′ were observed, in
addition to a weak correlation between H2′-H4′. Together with the observed coupling
constants, these NOE correlations indicate that H1′, H3′ and H5′ are in the axial positions and
confirm the β-D-glucose arrangement. The averaged chemical shift for the H6′ protons in 2
are 4.45 and 4.24 ppm, considerably different from the expected chemical shift of free
methylene protons at the C6′ position of β-D-glucose (3.25 ppm). Specific NOE correlations
were observed between (i) HN of Ser-10 and H6′ and (ii) the Ser-10 Hα proton resonance and
H6′ of the glucose. These correlations demonstrate that the C-terminal Ser residue of the C
10
peptide is attached on the C6′ position of the glucose moiety in 2. This arrangement is in full
accord with the assigned ester connectivity in MccE492m.
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Page 12
The chemical shifts for the H1′, H2′, H3′ and H5′ protons of the glucose in 1 are comparable
to those in 2. In contrast, the proton resonances and NOE correlations for H4′ and H6′ differ
1
in the NOESY and COSY spectra. Using the H-COSY spectra to assign the protons of the
sugar moiety in 1, we assert that the chemical shift for H4′ is 4.96 ppm, whereas the chemical
shift of H6′ is 3.98 ppm. With respect to 2, this is a flip-like change of the chemical shifts for
H4′ and H6′. The J-coupling constants and observed intra-moiety NOEs support the
configuration of the sugar in 1 as a β-D-glucose. Furthermore for 1, new NOE correlations
were identified between H4′ and one of the Hβ protons of Ser-10 and between H4′ and HN of
Gly-9 and H3′ and HN Ser-10. The inter-residual NOE contacts to H6′ observed in 2 could not
be identified for 1. These data indicate that the C-terminal serine residue of the C peptide is
10
attached to the C4′ position in the MceIJ-generated product.
The structural analysis and activity assays establish that MceIJ installs C onto the C4′ oxygen
10
of MGE via an ester linkage from Ser-10 and subsequent migration of C to the C6′ oxygen
10
moiety is enzyme-independent and occurs in the presence of a base to yield the connectivity
observed in a sample of MccE492m that was isolated from bacterial cultures and structurally
characterized (Scheme 1). The former observation was contrary to our expectation that MceIJ
would link C to the C6′ position of the MGE moiety. We rationalize the migration of the
10
decapeptide from C4′ to C6′ with a six-membered intermediate where the deprotonated C6′
hydroxyl attacks the carbonyl of the ester bond between Ser-10 and the C4′ hydroxyl (Scheme
1). This intermediate was previously suggested in the base-dependent migration of simple acyl
52
groups from C4′ to C6′ of glucose and analogous transformations have been reported for
53–56
acetylated pyranoses, including 1,2,3,4-tetra-O-acetyl-α-D-glucopyranose,
and
suggested in the isomerization of 1-O-indol-3-ylacetyl-β-D-glucose, an ester conjugate of a
57
plant growth hormone. These observations raise the question of whether other natural
products containing ester linkages at the C6′ position of glucose were first generated
enzymatically with alternative connectivity.
Further Considerations Regarding MccE492m Maturation
Having established that MceIJ can transfer MGE to the terminal serine residues of C and
10
MccE492, we questioned if lin-MGE is also a substrate. Incubation of MceIJ with C , lin-
10
MGE, ATP and MgCl at pH 8 resulted in the formation of three new peaks with catecholate
2
absorption in the analytical HPLC trace (Figure S5). Isolation and mass spectral analysis
indicated that each product has a molecular weight equivalent to C +(lin-MGE)-H O and thus
10
2
+
connection of C to lin-MGE through an ester linkage ([M+H] m/z calc 1658.5; found 1658.1,
10
1658.4, 1658.0). We presume that peak 2 (Figure S5) corresponds to the C -C4′-(lin-MGE)
10
isomer and peaks 3 and 4 (Figure S5) to structural isomers, one of which is C -C6′-(lin-MGE),
10
but NMR structural analysis is required to validate this notion.
With knowledge that MceIJ accepts both lin-MGE and MGE, we also sought to ascertain if
MceD recognizes the C -MGE products from MceIJ-catalyzed reactions. Incubation of C -
10
10
C6′-MGE with MceD at pH 7.5 for 2 min resulted in formation of a new peak in the analytical
HPLC trace with a mass consistent with hydrolysis of the MGE lactone ring and formation of
C -C6′-(lin-MGE) ([M+H] m/z calc, 1658.5; found, 1659.1) (Figure S6). The product
10
distribution at longer time points resembles that of MceD-catalyzed MGE hydrolysis (Figure
5) with initial build-up of the linear product and then further degradation (Figure S6). MS
analysis confirmed formation of the C -linked monoglycosylated DHB-Ser dimer ([M+H]
10
m/z calc, 1436.5; found, 1435.8) and monomer ([M+H] m/z calc, 1212.4; found, 1212.7). MceD
is therefore selective for hydrolysis of the macrolactone over hydrolysis of the ester linkage
between C and the glucose moiety. The C -C4′-MGE enzymatic product is also recognized
10
10
and processed similarly (data not shown). The HPLC assays suggest that some migration of
the C peptide from C4′ to the C6′ position occurs in the MceD-generated product during the
10
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Nolan et al.
Page 13
course of the reaction. Kinetic characterization of MceD-catalyzed hydrolysis of both C -
10
MGE isomers was therefore conducted (Table 2, Figure S6). A comparison of k and K for
cat
m
MGE and its peptide-derivatized analogs reveals that the C peptide has negligible effect on
10
MceD activity.
We depict the maturation of MccE492m in Scheme 2. This process commences with C-
glycosylation of Ent by MceC. The resulting MGE is either attached to MceA, the peptide
precursor of MccE492, by MceIJ or hydrolyzed by MceD to yield lin-MGE. In the former case,
MceIJ attaches MceA to the C4′ hydroxyl of MGE and base-catalyzed migration of the glycosyl
ester to the C6′ position affords the MccE492m connectivity. MceD linearizes the lactone
scaffolds of these C -MGE conjugates and cleavage of the MceA precursor peptide occurs
10
58
during export, which together yield MccE492m. In the latter case, MceIJ attaches lin-MGE
to the MceA C-terminus, presumably at the C4′ position. Migration of the glycosyl ester to the
C6′ position and cleavage of MceA affords MccE492m, which is exported by MceGH. The
versatility of MceIJ and MceD points to a mechanism of efficient toxin assembly and enhanced
survival. Although not considered in Scheme 2, MccE492m analogs bearing the glycosylated
6,58
DHB-Ser dimer and monomer have been indentified from bacterial cultures.
These species
may arise from the MceD-catalyzed hydrolysis of C -(lin-MGE), as shown in this work.
10
MceIJ-catalyzed attachment of these hydrolysis products to the peptide C-terminus is another
possible route that merits consideration.
Summary and Perspectives
Bacteria elaborate peptide-based antibiotics during periods of stress to compete and survive in
the host environment. Some of these peptides, including the beta lactams of the penicillin/
cephalosporin classes and glycopeptides of the vancomycin class, are synthesized by
nonribosomal peptide synthetases. Others, including the microcin family, are produced by the
ribosome. Many microcin peptide scaffolds are subject to distinct post-translational
modifications that are required for their biological activity. Enzymes co-expressed with the
structural gene in the microcin cluster catalyze these modifications. The post-translational
tailoring of MccE492 is noteworthy in several respects. Structurally, modification of the C-
terminal residue of a protein with an ester linkage to a glucose moiety is novel. Because the
glucose is also connected to enterobactin by a C-glycosidic bond, it bridges the ribosomal and
nonribosomal peptide fragments of MccE492m and thereby joins two elements with distinct
function. The lin-MGE added to MccE492 during maturation, formed by glycosylation of
enterobactin, constitutes a second layer of secondary metabolism. Subsequent attachment of
MccE492 to lin-Ent may be therefore viewed as a third layer of secondary metabolism and
further exemplifies Nature’s biosynthetic ingenuity. Further study of the MceIJ-catalyzed
attachment of MGE to the MccE492 C-terminus is particularly warranted for mechanism and
selectivity.
From a functional standpoint, maturation of MccE492m creates a Trojan horse toxin directed
to virulent bacteria that express catecholate siderophore receptors. Other toxic natural products
exploit this type of recognition strategy. Albomycin, produced by Streptomyces, is comprised
of a nucleoside antibiotic linked to a hydroxamate siderophore and it binds siderophore-specific
59–61
62
63
porins,
as does Colicin Ia. Siderophore uptake pumps also grant cell entry to larger
cargo, such as bacteriophage H8, and have been considered as targets for combating
Pseudomonas infections.
64,65
This siderophore-based approach to cell penetration may be
applicable in the design of new antibiotics that use iron-scavengers to target specific microbial
populations, including gram-negative and multi-drug resistant strains.
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Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
Acknowledgements
This work was supported by NIH Grant AI47238 (C.T.W.). NMR instrumentation was supported by Grant GM47467
from the National Institute of General Medical Sciences to Prof. Gehard Wagner. We thank Prof. Rosalba Lagos,
Claudia Estevez and Mario Tello for a gift of the pJEM15 plasmid and for insightful discussions. We also thank Carl
Balibar and Jay Read for helpful suggestions. EMN acknowledges the NIH for a post-doctoral fellowship, MAP thanks
the Hertz Foundation for a graduate fellowship and AK is a recipient of a long-term fellowship from the Human
Frontier Science Program Organization.
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Figure 1.
Structures of enterobactin (Ent), linearized enterobactin (lin-Ent), monoglycosylated
enterobactin (MGE), linearized MGE (lin-MGE) and a truncated depiction of microcin E492m
(MccE492m) showing the last eleven amino acids of the microcin peptide. Diglycosylated
(DGE) and triglycosylated enterobactin (TGE) have glucose moieties attached to the C5
positions of either two or three DHB-Ser units, respectively.
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Figure 2.
(a) The MccE492 gene cluster: mceA, 312 bp; mceB, 288 bp; mceC, 1113 bp; mceD, 1245 bp;
mceE, 345 bp; mceF, 540 bp; mceG, 2097 bp; mceH, 1242 bp; mceI, 492 bp; mceJ, 1575 bp
(ref. 1). (b) Amino acid sequences for the serine-rich C-termini of MccE492, MccH47, MccI47
and MccM. The sequence of the C-terminus of MccE492 was determined experimentally.
MccH47, MccI47 and MccM have not been isolated and their C-terminal sequences were
deduced from the respective genes.
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Figure 3.
SDS-Page gel (4–15% Tris-HCl) of purified His fusions of MceC (1, C-terminal His fusion),
6
6
MceD (2, C-terminal His fusion; 3, N-terminal His fusion) and MceIJ (4, MceJ bears a N-
6
6
terminal His tag).
6
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Figure 4.
MceC C-glycosylates Ent. (a) Representation of the MceC-catalyzed converstion of Ent to
MGE and DGE. (b) HPLC analysis of the MceC-catalyzed glycosylation of Ent. (c) Kinetic
traces for MceC-catalyzed glycosylation of Ent, MGE and lin-Ent. The corresponding k and
cat
K values are listed in Table 1.
m
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Figure 5.
MceD hydrolyzes apo Ent, MGE and DGE. (a) HPLC analysis of MceD-catalyzed hydrolysis
of Ent. Dimer and monomer refer to the DHB-Ser dimer and DHB-Ser monomer, respectively,
(b) HPLC analysis of MceD-catalyzed hydrolysis of MGE. Glc-Dimer is the monoglycosylated
DHB-Ser dimer and “Monomers” refer to the Glc-DHB-Ser (elution time ~ 2 min) and DHB-
Ser (elution time ~2.5 min) monomers, the former of which results from hydrolysis of the Glc-
DHB-Ser dimer. (c) Kinetic traces for the MceD-catalyzed hydrolysis of Ent, lin-Ent, MGE,
lin-MGE, and DGE. Corresponding kinetic parameters are listed in Table 2. All reactions were
carried out at room temperature (75 mM Hepes, pH 7.5). (d) Schematic of the MceD-catalyzed
hydrolysis of MGE illustrating the regioselectivity of the cut as determined by 2-D NMR.
HPLC traces for assays with DGE are given in Figure S2 and the m/z data for Ent, MGE and
DGE hydrolysis products are listed in Table S1.
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Figure 6.
HPLC monitoring of a reaction containing 550 μM C , 100 μM MGE and 2 μM MceIJ, 5 mM
10
ATP and 5 mM MgCl (75 mM Tris-HCl pH 8, 2.5 mM TCEP). The two new peaks, 1 and
2
2, both have a mass equivalent to C +MGE-H O, which suggests attachment of MGE to the
10
2
C
peptide through an ester linkage.
10
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Figure 7.
1
(a) Section of the 2D- H homonuclear NOESY spectra for C -C4′-MGE (peak 1). (b) Section
of the 2D- H homonuclear NOESY spectra for C -C6′-MGE (peak 2). Both spectra were
10
1
10
acquired using a W5 pulse sequence for water suppression and a 250 msec mixing time. The
dotted lines indicate the positions of the sugar protons and the corresponding NOE correlations.
The proton assignments are listed in Table S2. (c) Analytical HPLC (220 nm absorption) of
purified C -C6′-MGE. (d) Analytica HPLC trace (220 nm absorption) of purified C -C4′-
10
10
MGE. A gradient of 0 to 40% MeCN in 8 min was employed for both samples, (e) Numbering
scheme for the glucose and DHB moieties.
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Scheme 1.
(a) Attachment of MGE to the MccE492m C-terminal decapeptide (C ) by MceIJ through the
10
C4′ hydroxyl and subsequent migration to the C6′ position, (b) Formation of the six-membered
intermediate between the C4′ and C6′ positions.
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Scheme 2.
Maturation of MccE492m as defined by in vitro studies. MceA is the peptide precursor to
MccE492. It undergoes cleavage at amino acid 15 or 19 during export to yield the 84-residue
MccE492(m).
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