The nonribosomal peptide synthetase enzyme DdaD tethers N β-fumaramoyl-l -2,3-diaminopropionate for Fe(II)/α- ketoglutarate-dependent epoxidation by DdaC during dapdiamide antibiotic biosynthesis

Article abstract of DOI:10.1021/ja1072367

The gene cluster from Pantoea agglomerans responsible for biosynthesis of the dapdiamide antibiotics encodes an adenylation-thiolation didomain protein, DdaD, and an Fe(II)/α-ketoglutarate-dependent dioxygenase homologue, DdaC. Here we show that DdaD, a nonribosomal peptide synthetase module, activates and sequesters N_β-fumaramoyl-l-2,3-diaminopropionate as a covalently tethered thioester for subsequent oxidative modification of the fumaramoyl group. DdaC catalyzes Fe(II)- and α-ketoglutarate-dependent epoxidation of the covalently bound N_β-fumaramoyl-l-2,3- diaminopropionyl-S-DdaD species to generate N_β-epoxysuccinamoyl- DAP (DAP = 2,3-diaminopropionate) in thioester linkage to DdaD. After hydrolytic release, N_β-epoxysuccinamoyl-DAP can be ligated to l-valine by the ATP-dependent ligase DdaF to form the natural antibiotic N _β-epoxysuccinamoyl-DAP-Val.

Full text of DOI:10.1021/ja1072367

Published on Web 10/14/2010
The Nonribosomal Peptide Synthetase Enzyme DdaD Tethers
Nꢀ-Fumaramoyl-L-2,3-diaminopropionate for Fe(II)/
r-Ketoglutarate-Dependent Epoxidation by DdaC during
Dapdiamide Antibiotic Biosynthesis
Marie A. Hollenhorst,† Stefanie B. Bumpus,‡ Megan L. Matthews,§
J. Martin Bollinger, Jr.,| Neil L. Kelleher,‡,⊥ and Christopher T. Walsh*,†
Department of Biological Chemistry and Molecular Pharmacology, HarVard Medical School,
Boston, Massachusetts 02115, Department of Chemistry and Institute for Genomic Biology,
UniVersity of Illinois at Urbana-Champaign, Urbana, Illinois 61801, and Departments of
Chemistry, Biochemistry, and Molecular Biology, The PennsylVania State UniVersity, UniVersity
Park, PennsylVania 16802, United States
Received August 11, 2010; E-mail: christopher_walsh@hms.harvard.edu
Abstract: The gene cluster from Pantoea agglomerans responsible for biosynthesis of the dapdiamide
antibiotics encodes an adenylation-thiolation didomain protein, DdaD, and an Fe(II)/R-ketoglutarate-
dependent dioxygenase homologue, DdaC. Here we show that DdaD, a nonribosomal peptide synthetase
module, activates and sequesters Nꢀ-fumaramoyl-L-2,3-diaminopropionate as a covalently tethered thioester
for subsequent oxidative modification of the fumaramoyl group. DdaC catalyzes Fe(II)- and R-ketoglutarate-
dependent epoxidation of the covalently bound Nꢀ-fumaramoyl-L-2,3-diaminopropionyl-S-DdaD species to
generate Nꢀ-epoxysuccinamoyl-DAP (DAP ) 2,3-diaminopropionate) in thioester linkage to DdaD. After
hydrolytic release, Nꢀ-epoxysuccinamoyl-DAP can be ligated to L-valine by the ATP-dependent ligase DdaF
to form the natural antibiotic Nꢀ-epoxysuccinamoyl-DAP-Val.
Introduction
The dapdiamides A-E were isolated by activity-based
cloning of dapdiamide biosynthetic genes from P. agglomerans
CU0119 into Escherichia coli.1 This allowed for sequencing
of the responsible gene cluster, which revealed nine genes,
annotated as ddaA-I (Figure 1B), that are necessary and
sufficient for E. coli to make the dapdiamides. In our initial
studies on the Dda enzymes, we determined that DdaG and
DdaF are ATP-dependent ligases that build the N-acyldipeptide
scaffolds.4 DdaG is an AMP-generating ligase that makes the
regiospecifically N-acylated Nꢀ-fumaroyl-DAP (NꢀFmDAP). Our
data indicated that DdaF catalyzes the last step in the pathway
and forms the dipeptide linkage of N-acyl-DAP with Val, Ile,
or Leu. DdaF cleaves ATP to ADP (not AMP), generating the
N-acyl-DAP-phosphate as an activated intermediate for capture
by Val, Ile, or Leu. This enzyme accepts only Nꢀ-fumaramoyl-
DAP (NꢀFmmDAP), not NꢀFmDAP, as the carboxylate sub-
strate, suggesting DdaH, the putative fumaroyl to fumaramoyl
amide synthase, acts after DdaG but before DdaF.
The dapdiamide antibiotics are a family of five N-acylated
dipeptides produced by Pantoea agglomerans (Figure 1A).1 The
“dap” prefix refers to the presence of the nonproteinogenic
amino acid 2,3-diaminopropionate (DAP; blue in Figure 1),
while the “diamide” suffix reflects the two backbone amide
bonds. The DAP moiety, which can be acylated either on Nꢀ
or on NR, is the first residue of the dipeptide and is attached via
a standard peptide linkage to a terminal valine (Val), isoleucine
(Ile), or leucine (Leu) (red in Figure 1). The N-acyl moiety
(green in Figure 1) is a fumaramoyl group in dapdiamides A-D
and an epoxysuccinamoyl group in dapdiamide E. The fuma-
ramoyl or epoxysuccinamoyl functionality most likely provides
the electrophilic moiety that accounts for the antibiotic activity
of this class of compounds.2 The dapdiamides are likely cleaved
intracellularly to generate acyl-DAP warheads that target
glucosamine-6-phosphate synthase via capture of the nucleo-
philic active site cysteine (Cys).2,3
In this study, we have turned our attention to the epoxidation
event in the dapdiamide biosynthetic pathway. An epoxysucci-
namoyl moiety is present in both dapdiamide E and its Nꢀ-acyl-
DAP isomer Nꢀ-epoxysuccinamoyl-DAP-Val (NꢀEpSmDAP-
Val) [this compound has been referred to in the literature
variously as CB-25-I, [2-amino-3-(oxirane-2,3-dicarboxami-
do)propanoyl]valine, and herbicolin I], a natural product
produced by Serratia plymuthica and P. agglomerans strains
† Harvard Medical School.
‡ University of Illinois at Urbana-Champaign.
§ Department of Chemistry, The Pennsylvania State University.
| Departments of Chemistry, Biochemistry, and Molecular Biology, The
Pennsylvania State University.
⊥ Current address: Department of Chemistry, Northwestern University,
Evanston, IL 60208, United States.
(1) Dawlaty, J.; Zhang, X.; Fischbach, M. A.; Clardy, J. J. Nat. Prod.
2010, 73, 441–446.
(2) Kucharczyk, N.; Denisot, M. A.; Le Goffic, F.; Badet, B. Biochemistry
1990, 29, 3668–3676.
(3) Milewski, S.; Andruszkiewicz, R.; Kasprzak, L.; Mazerski, J.; Mignini,
F.; Borowski, E. Antimicrob. Agents Chemother. 1991, 35, 36–43.
(4) Hollenhorst, M. A.; Clardy, J.; Walsh, C. T. Biochemistry 2009, 48,
10467–10472.
9
10.1021/ja1072367  2010 American Chemical Society
J. AM. CHEM. SOC. 2010, 132, 15773–15781 15773

A R T I C L E S
Hollenhorst et al.
However, there are other known natural products that contain
both (R,R)- and (S,S)-trans-epoxysuccin(am)ate.9,10
Here we report that DdaF catalyzes ATP-dependent ligation
of both of the Nꢀ-trans-epoxysuccinamoyl-DAP (Nꢀ-trans-
EpSmDAP) diastereomers and Val, with substrate specificity
for the (R,R) over the (S,S) diastereomer. Our studies were based
on the hypothesis that DdaC and DdaD are required to form
Nꢀ-trans-EpSmDAP from an olefin-containing acyl-DAP inter-
mediate. DdaD is a third type of ATP-utilizing enzyme in this
pathway, a predicted nonribosomal peptide synthetase (NRPS)
module,11 while DdaC has homology to mononuclear nonheme
iron oxygenases.12 In this study, we demonstrate that DdaC and
DdaD are the relevant catalysts and determine the timing of
epoxidation during Nꢀ-acyl-DAP-Val assembly (Scheme 1).
Materials and Methods
Materials and General Methods. Oligonucleotide primers were
synthesized by Integrated DNA Technologies (Coralville, IA).
Polymerase chain reaction (PCR) was performed with Phusion
High-Fidelity PCR Mastermix (New England Biolabs). Cloning was
performed using the Gateway System (Invitrogen). One Shot
Chemically Competent TOP10 E. coli (Invitrogen) and Nova-
Blue(DE3) (Novagen) were used for routine cloning and propaga-
tion of DNA vectors. Recombinant plasmid DNA was purified with
a Qiaprep kit (Qiagen). DNA sequencing was performed at the
Molecular Biology Core Facilities of the Dana Farber Cancer
Institute (Boston, MA). Nickel-nitrilotriacetic acid agarose
(Ni-NTA) superflow resin and sodium dodecyl sulfate-polyacryl-
amide gel electrophoresis (SDS-PAGE) gels were purchased from
Qiagen and Biorad, respectively. Protein concentrations were
determined by Bradford assay13 with bovine serum albumin (BSA)
as a standard or with a Nanodrop 1000 spectrophotometer (Thermo
Scientific) on the basis of the absorbance at 280 nm with the
predicted molar extinction coefficient.
Anaerobic manipulations were performed under a nitrogen
atmosphere using an Mbraun Labmaster glovebox (Stratham, NH)
maintained at 2 ppm O2 or less. Buffers were sparged with argon
for 20-30 min and equilibrated overnight with the nitrogen
atmosphere in the glovebox before use.
A pyruvate kinase/lactate dehydrogenase (PK/LDH) enzyme mix
from rabbit muscle was purchased from Sigma as a buffered
aqueous glycerol solution. Synthetic dapdiamide A and the plasmid
containing the dapdiamide gene cluster, pUC19 A10A, were
provided by Jessica Dawlaty (Harvard Medical School, Boston,
MA).1 BODIPY-CoA14 and Sfp15,16 were prepared according to
published procedures. N-His6-tagged DdaF was purified as described
previously.4 NꢀFmDAP, NRFmDAP, NꢀFmmDAP, NRFmmDAP,
and fumaramate (Fmm) were synthesized as described previously.4
DdaF ADP Production Assay. The reaction mixtures (250 µL)
were incubated at room temperature and contained 250 nM DdaF,
5 mM Val, 10 mM ATP, 12 mM MgCl2, 200 µM NADH, 500 µM
phosphoenolpyruvate, 59 units/mL pyruvate kinase, 41 units/mL
lactate dehydrogenase, 5 mM borate ·NaOH (pH 9.5), and 100 mM
4-(2-hyhdroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (pH
8). The consumption of NADH was monitored continuously in
Figure 1. (A) Dapdiamide family of antibiotics. (B) Dapdiamide gene
cluster. (C) Dapdiamide-related epoxide natural products.
48b/90, 39b/90, and C9-1 (Figure 1C).1,5,6 NMR evidence
suggests that each of these compounds contains a trans-epoxide,
but the absolute stereochemistry of the epoxide carbons has not
been determined.1,5,6 The most similar epoxysuccin(am)ate-
containing natural product for which the epoxide stereochemistry
has been determined is Sch37137 (Figure 1C), and in this
compound the epoxide carbons have an (R,R) configuration.7,8
(9) Martin, W. R.; Foster, J. W. J. Bacteriol. 1955, 70, 405–414.
(10) Hanada, K.; Tamai, M.; Ohmura, S.; Sawada, J.; Seki, T.; Tanaka, I.
Agric. Biol. Chem. 1978, 42, 529–536.
(5) Shoji, J.; Hinoo, H.; Sakazaki, R.; Kato, T.; Hattori, T.; Matsumoto,
K.; Tawara, K.; Kikuchi, J.; Terui, Y. J. Antibiot. 1989, 42, 869–874.
(6) Sammer, U. F.; Volksch, B.; Mollmann, U.; Schmidtke, M.; Spiteller,
P.; Spiteller, M.; Spiteller, D. Appl. EnViron. Microbiol. 2009, 75,
7710–7717.
(11) Fischbach, M. A.; Walsh, C. T. Chem. ReV. 2006, 106, 3468–3496.
(12) Hausinger, R. P. Crit. ReV. Biochem. Mol. Biol. 2004, 39, 21–68.
(13) Bradford, M. M. Anal. Biochem. 1976, 72, 248–254.
(14) La Clair, J. J.; Foley, T. L.; Schegg, T. R.; Regan, C. M.; Burkart,
M. D. Chem. Biol. 2004, 11, 195–201.
(7) Cooper, R.; Horan, A. C.; Gentile, F.; Gullo, V.; Loebenberg, D.;
Marquez, J.; Patel, M.; Puar, M. S.; Truumees, I. J. Antibiot. 1988,
41, 13–19.
(15) Quadri, L. E. N.; Weinreb, P. H.; Lei, M.; Nakano, M. M.; Zuber, P.;
Walsh, C. T. Biochemistry 1998, 37, 1585–1595.
(16) Lambalot, R. H.; Gehring, A. M.; Flugel, R. S.; Zuber, P.; LaCelle,
M.; Marahiel, M. A.; Reid, R.; Khosla, C.; Walsh, C. T. Chem. Biol.
1996, 3, 923–936.
(8) Rane, D. F.; Girijavallabhan, V. M.; Ganguly, A. K.; Pike, R. E.;
Saksena, A. K.; McPhail, A. T. Tetrahedron Lett. 1993, 34, 3201–
3204.
9
15774 J. AM. CHEM. SOC. VOL. 132, NO. 44, 2010

Epoxidation in Dapdiamide Biosynthesis
A R T I C L E S
Scheme 1. Proposed Role of DdaC-F in the Formation of NꢀEpSmDAP-Vala
a The grayed-out step has not been biochemically validated.
Plastibrand micro UV cuvettes in a Varian Cary 50 UV-vis
spectrophotometer by measuring the absorbance at 340 nm. Kinetic
constants were derived from velocity versus substrate concentration
data with GraphPad Prism using a nonlinear, least-squares fitting
method for NꢀFmmDAP and Nꢀ-(R,R)-epoxysuccinamoyl-DAP
(NꢀRREpSmDAP) and a linear regression for Nꢀ-(S,S)-epoxysuc-
cinamoyl-DAP (NꢀSSEpSmDAP).
diamide A onto HS-DdaD, and (6) incorporation of 18O into
NꢀEpSmDAP-S-DdaD using H218O.
All reactions were stored at -80 °C until trypsin digestion. Prior
to trypsin digestion, the samples were thawed at room temperature.
Trypsin (Promega Sequencing grade) was resuspended in the buffer
provided by the manufacturer to a final concentration of 1 µg/µL
and incubated at 30 °C for 15 min. An aliquot of the reaction
containing 50 µg of DdaD was removed and added to an equal
volume of 0.1 M NH4HCO3 (pH 7.8-8) and 2 mM Tris(2-
carboxyethyl)phosphine (TCEP) (pH 6). This mixture was incubated
for 4 min at room temperature, and trypsin was added at a mass
ratio of 1:5 trypsin:total protein in the digestion reaction. After
addition of the trypsin, the reaction was incubated at 30 °C for 5
min. The reaction was quenched by the addition of one-half reaction
volume of 25% formic acid and stored at -80 °C until mass
spectrometric analysis.
RPLC-FTMS Analysis of Trypsin Digests. All RPLC-FTMS
analyses were conducted using an Agilent 1200 high-performance
LC (HPLC) system with an autosampler coupled directly to a
ThermoFisher Scientific LTQ-FT hybrid linear ion trap FTMS
system operating at 11 T. The mass spectrometer was calibrated
weekly using the calibration mixture and instructions specified by
the manufacturer. All instrument parameters were tuned according
to the manufacturer’s instructions (employing bovine ubiquitin
(Sigma) for tuning purposes).
For all analyses of trypsin digests of DdaD-containing reactions,
a 1 mm × 150 mm Jupiter C18 column (Phenomenex, 300 Å, 5
µm) was connected in-line with the electrospray ionization (ESI)
source (operated at ∼5 kV with a capillary temperature of 200-250
°C) for the MS system. The 70 min separation gradient used for
all RPLC analyses is shown in Table S1 (Supporting Information),
where solvent A was H2O + 0.1% formic acid and solvent B was
acetonitrile (MeCN) + 0.1% formic acid. A trypsin-digested
reaction mixture was loaded onto the column using the autosampler
and separated according to the gradient shown.
All ionized peptide species entering the mass spectrometer were
subjected to an MS method with five MS and MS/MS events: (1)
full scan measurement of all intact peptides (all ions detected in
the FTMS instrument in profile mode, resolution 100 000, m/z range
detected 400-2000), (2) the phosphopantetheinyl (Ppant) ejection
assay using nozzle-skimmer dissociation (NSD) (all ions detected
in the FTMS instrument in profile mode, resolution 50 000, m/z
range 250-500, surface-induced dissociation (SID) 75 V), (3-5)
data-dependent MS/MS on the first, second, and third most abundant
ions from scan 1 using collision-induced dissociation (CID) (all
ions detected in the FTMS instrument in profile mode, minimum
target signal counts 5000, resolution 50 000, m/z range detected
dependent on target m/z, default charge state 2, isolation width 5
DdaD ATP-[32P]PPi Exchange Assays. The reaction mixtures
(170-350 µL) contained 5 µM DdaD, 125 µM substrate, 1 mM
ATP, 1 mM MgCl2, 40 mM KCl, 5 mM Na[32P]PPi (approximately
(2-4) × 107 counts per min (cpm)/mL), and 50 mM HEPES (pH
7.5). The reactions were incubated at room temperature for 10 min,
and then 50 µL aliquots were removed and quenched with 250 µL
of a charcoal suspension (100 mM NaPPi, 350 mM HClO4, and 16
g/L charcoal). The samples were mixed using a vortex and then
centrifuged at 16000g for 3 min. The pellets were washed twice
with 250 µL of wash solution (100 mM NaPPi, and 350 mM
HClO4). Charcoal-bound radioactivity was measured on a Beckman
LS 6500 scintillation counter.
DdaC/D Enzymatic Assays with Anaerobically Purified
DdaC. Phosphopantetheinylation reactions (12-270 µL) contained
25 µM DdaD, 12 µM Sfp, 400 µM coenzyme A (CoA), 10 mM
MgCl2, 1.5 mM dithiothreitol (DTT), and 50 mM HEPES (pH 7.5).
The reactions were incubated at room temperature for 1 h. To form
aminoacyl-S-DdaD, 120-250 µM substrate and 1 mM ATP were
added (final volume 12.5-156 µL) and the reactions incubated for
an additional 5-60 min at room temperature.
The following reagents were added to the NꢀFmmDAP-S-DdaD
preparations to the indicated final concentrations: 50 µM
Fe(NH4)2(SO4)2 (stock solution 2.5 mM in 200 µM HCl), 300 µM
R-ketoglutarate (R-KG) (stock solution 6 mM in 50 mM HEPES
(pH 8)), 4 mM ascorbic acid (stock solution 100 mM in 50 mM
HEPES (pH 8)), and 10 µM DdaC (final volume 73-79 µL). The
reactions were incubated for 5-60 min at room temperature.
The reactions were quenched by flash freezing in N2(l) for
subsequent trypsin digestion or by the addition of an equal volume
of 25% formic acid followed by flash freezing in N2(l) for reversed-
phase liquid chromatography (RPLC)-Fourier transform mass
spectrometry (FTMS) analysis of intact DdaD.
H218O incubations were carried out as described above with the
exception that the final reaction mixtures contained 63% H218O
(Cambridge Isotope Laboratories).
Trypsin Digestion of DdaD-Containing Reactions. The fol-
lowing digestion procedure was used for analysis of (1) conversion
of apo-DdaD to holo-DdaD (HS-DdaD), (2) loading of DdaD with
NꢀFmmDAP to generate NꢀFmmDAP-S-DdaD, (3) conversion of
NꢀFmmDAP-S-DdaD to NꢀEpSmDAP-S-DdaD by DdaC, (4)
dependence of epoxide formation on R-KG, (5) loading of dap-
9
J. AM. CHEM. SOC. VOL. 132, NO. 44, 2010 15775

A R T I C L E S
Hollenhorst et al.
Table 1. DdaF Kinetics with Respect to NꢀFmmDAP and
m/z, normalized collision energy (NCE) 35, activation q value 0.40,
activation time 30 ms). During all analyses, dynamic exclusion was
enabled with the following settings: repeat count 2, repeat duration
30 s, exclusion list size 300, exclusion duration 60 s.
NꢀEpSmDAPsa
substrate
kcat (min-1
)
Km (µM)
kcat/Km (min-1 mM-1
)
NꢀFmmDAP
21 ( 3
72 ( 34
53 ( 7
ND
290
3400
82 ( 8
All data were analyzed using Qualbrowser (Xcalibur) and
ProSightPC, both provided with the LTQ-FT system. ProsightPC
was used to search all MS/MS data against a database containing
the apo-DdaD protein sequence for peptide identification, with an
intact peptide mass tolerance of 750 Da (which allowed for
observation of HS-DdaD and HS-DdaD loaded with a variety of
substrates) and a fragment ion mass tolerance of 10 ppm (the ∆m
mode was enabled for all searches). A minimum of five matching
fragment ions were required for peptide identification. In Qual-
browser, ions of interest were searched within a range of 0.01 m/z
around the isotopic peak of interest, within a tolerance of 5 ppm.
RPLC-FTMS Analysis of Intact DdaD. RPLC-FTMS analy-
sis of intact DdaD by the Ppant ejection assay was employed for
analyses of the loading of NꢀRREpSmDAP onto HS-DdaD, the
loading of NꢀSSEpSmDAP onto HS-DdaD, and 18O incorporation
into NꢀEpSmDAP-S-DdaD using 18O2. All reactions were quenched
with an equal volume of 25% formic acid and stored at -80 °C
prior to analysis.
For all analyses of intact proteins in DdaD-containing reactions,
a 1 mm ×150 mm Jupiter C4 column (Phenomenex, 300 Å, 5 µm)
was connected in-line with the ESI source (operated at ∼5 kV with
a capillary temperature of 200-250 °C) for the MS system. A
reaction aliquot containing 12-15 µg of DdaD was injected for
each sample. The 45 min separation gradient used for all RPLC
analyses is shown in Table S2 (Supporting Information), where
solvent A was H2O + 0.1% formic acid and solvent B was MeCN
+ 0.1% formic acid. The reaction mixture was loaded onto the
column using the autosampler and separated according to the
gradient shown.
All ionized protein species entering the mass spectrometer were
subjected to an MS method with two MS and MS/MS events: (1)
full scan measurement of all intact peptides (all ions detected in
the ion trap MS instrument in profile mode, m/z range detected
400-2000), (2) Ppant ejection assay using NSD (all ions detected
in the FTMS instrument in profile mode, resolution 50 000, m/z
range 250-500, SID ) 75 V). All data were analyzed using
Qualbrowser (Xcalibur), provided for analysis with the LTQ-FT
system. Ppant ejection ions of interest were searched within a range
of 0.01 m/z around the isotopic peak of interest, within a tolerance
of 5 ppm.
Determination of the Source of the Epoxide Oxygen by Use
of 18O2(g). Phosphopantetheinylation reactions (506 µL) contained
25 µM DdaD, 12 µM Sfp, 400 µM CoA, 1 mM MgCl2, 1.5 mM
DTT, and 50 mM HEPES (pH 7.5). The reactions were incubated
at room temperature for 1 h. To form NꢀFmmDAP-S-DdaD, 123
µM NꢀFmmDAP and 5.5 mM ATP were added (final volume 522
µL) and the reactions incubated for an additional 5 min at room
temperature.
NꢀRREpSmDAP
181 ( 7
NꢀSSEpSmDAPb
ND
a The values were determined by coupled spectrophotometric ADP
production assay with a fixed Val concentration of 5 mM. Data are
presented as the value ( standard deviation. ND ) not determined.
b Saturation not achieved at concentrations up to 590 µM.
Nucleotide Sequence Accession Number. The nucleotide
sequence of the dapdiamide gene cluster from P. agglomerans strain
CU0119 has been deposited in the NCBI GenBank database under
accession number HQ130277.
Results
DdaF Ligates NꢀEpSmDAP and Val in an ATP-Dependent
Manner. Guided by the hypothesis that the dapdiamide pathway
produces an antibiotic containing a trans-epoxysuccinamate
moiety, we synthesized both diastereomers of Nꢀ-trans-EpSm-
DAP (see the Supporting Information for synthetic protocols
and characterization) and tested the activity of DdaF in ligating
these compounds to Val.
Analysis of the enzymatic assays by LC-MS (see the
Supporting Information for the method) revealed that DdaF
can catalyze the ligation of both Nꢀ-trans-EpSmDAP dia-
stereomers to Val to produce the NꢀEpSmDAP-Val dipeptide
antibiotics (Figure S1, Supporting Information). A coupled
spectrophotometric ADP production assay was used to
kinetically characterize the activity of DdaF with respect to
the two Nꢀ-trans-EpSmDAP diastereomers. DdaF was found
to use NꢀRREpSmDAP as a saturable substrate, whereas
saturation was not achieved with NꢀSSEpSmDAP at concentra-
tions up to 590 µM (Table 1; Figure S2, Supporting Informa-
tion). These findings suggested that NꢀRREpSmDAP may be
an on-pathway intermediate, with DdaF catalytic efficiency for
this substrate approximately 40-fold greater than that for the
corresponding (S,S)-epoxide.
Expression and Purification of DdaC and DdaD in E. coli.
The ddaC and ddaD genes were amplified from pUC19 A10A,
a plasmid containing the dapdiamide gene cluster,1 and cloned
into an expression vector encoding an N-terminal His6 tag for
DdaC or a C-terminal His6 tag for DdaD. The proteins were
overexpressed in E. coli BL21(DE3) and purified by Ni-NTA
affinity chromatography (see the Supporting Information for the
methods, Figure S3). Yields ranged from 4 to 6 mg/L for DdaC
and from 11 to 14 mg/L for DdaD.
O2-free solutions (prepared as previously described17) (225 µL)
containing NꢀFmmDAP-S-DdaD and R-KG (0.13 mM) in the
absence or presence of DdaC (4.2 µM) were placed in septum-
sealed flasks. The flasks were stirred on ice and briefly evacuated
to ∼30 Torr. One flask was subsequently refilled with 1.3 atm of
a gas mixture containing 80% 18O2(g) (95-98% isotopic enrich-
ment) and 20% N2(g), and the second flask was refilled with 1.1
atm of natural-abundance O2(g). Fe(NH4)2(SO4)2 and ascorbic acid
were subsequently added to the flasks (to concentrations of 21 µM
and 1.7 mM, respectively) via a gastight syringe. The solutions
were stirred for 5 min at 0 °C to allow the reactions to reach
completion. The flasks were opened to air, the reactions were
terminated by addition of each solution to an equal volume of 25%
formic acid, and the acidified mixtures were flash frozen in N2(l).
Following Ni-NTA chromatography, DdaC was either flash
frozen to yield an aerobic preparation or gel filtered into an
anaerobic atmosphere. The anaerobic preparations were incu-
bated with Fe(NH4)2(SO4)2, R-KG, and DTT and subsequently
desalted, giving a preparation with an iron occupancy of 47 (
4% (average ( standard deviation, data from two independent
experiments) by ferene spectrophotometric assay.18
DdaD Activates and Covalently Tethers NꢀFmmDAP. The
substrate specificity of the DdaD adenylation (A) domain was
probed using ATP-[32P]PPi exchange assays,19 which revealed
that NꢀFmmDAP is the preferred substrate (Figure 2). The
kinetics of NꢀFmmDAP adenylation by DdaD were determined
(18) Hennessy, D. J.; Reid, G. R.; Smith, F. E.; Thompson, S. L. Can.
J. Chem. 1984, 62, 721–724.
(17) Price, J. C.; Barr, E. W.; Tirupati, B.; Bollinger, J. M., Jr.; Krebs, C.
Biochemistry 2003, 42, 7497–7508.
(19) Lee, S. G.; Lipmann, F. Methods Enzymol. 1975, 43, 585–602.
9
15776 J. AM. CHEM. SOC. VOL. 132, NO. 44, 2010

Epoxidation in Dapdiamide Biosynthesis
A R T I C L E S
domain tryptic peptide that underwent the expected 340.09 Da
mass shift for PPTation (Figure 3A,B; Table S3, Supporting
Information). The PPTated peptide was subjected to two
different types of MS/MS fragmentation; NSD resulted in the
formation of the predicted small molecule Ppant ejection product
(a 1+ ion at m/z 261.127) (Figure 3C, Table S3), and CID
resulted in the formation of the predicted Ppant ejection peptide
marker ions (Figure 3D). These are the species corresponding
to apo-DdaD - 18 Da, from the formation of dehydroalanine
(Dha) at the active site Ser (brown in Figure 3D, 2+ ion at m/z
1451.6994, with an intact mass of 2901.3828 Da, an error of
-7 ppm from the theoretical mass), and to apo-DdaD + 80
Da, from the retention of the phosphate moiety on the active
site Ser during loss of the small molecule ejection ion with m/z
261.127 (tan in Figure 3D, 2+ ion at m/z 1500.7010, with an
intact mass of 2999.386 Da, an error of 1 ppm from the
theoretical mass). Localization of the site of Ppant modification
was achieved by CID MS/MS fragmentation of the PPTated T
domain active site tryptic peptide. This analysis demonstrated
that Ser533, which aligns with known T domain active site Ser
residues, is indeed the site of PPTation (Figure S7, Supporting
Information).
When DdaD, ATP, and NꢀFmmDAP were incubated, we
observed loading of the substrate onto the T domain both by
the mass shift of the intact peptide (a mass shift of +183.066
Da from the HS-DdaD active site peptide, where the theoretical
mass shift is +183.064 Da) and by Ppant ejection to produce
an ion at m/z 444.2 (Figure 3E-G; Table S3, Supporting
Information). Additional confirmation that NꢀFmmDAP is
covalently tethered to the DdaD T domain came from LC-MS
analysis of the NꢀFmmDAP compound following nonenzymatic
hydrolysis from the T domain (Figure S8, Supporting Informa-
tion). Using LC-MS and the Ppant ejection assay to analyze
intact DdaD, we also found that DdaD is capable of loading
both diastereomers of Nꢀ-trans-EpSmDAP (Figure S9, Sup-
porting Information). We did not observe any loading of
dapdiamide A (Figure S10, Supporting Information).
Figure 2. DdaD ATP-[32P]PPi exchange data. In these experiments, DdaD,
[32P]PPi, unlabeled ATP, and substrate were incubated, and then ATP
separated from PPi by its specific adsorption to charcoal. The graph shows
counts per minute arising from scintillography of the charcoal. (A)
Adenylation activity with a variety of potential substrates. The reactions
were quenched after 10 min. (B) Time course with NFmmDAPs,
NꢀEpSmDAPs, and dapdiamide A.
by this assay; the Km value is 420 ( 80 µM, and the kcat value
is 64 ( 5 min-1 (Figure S4, Supporting Information). DdaD
was also active with NꢀSSEpSmDAP but to a much lesser extent,
with a kcat/Km of 2.3 min-1 mM-1 compared with 150 min-1
mM-1 for NꢀFmmDAP (Figure S4). No exchange was observed
with NꢀRREpSmDAP.
The competence of the DdaD T domain to be phosphopanteth-
einylated (PPTated) was initially validated by the observation that
it could be modified by the promiscuous Bacillus subtilis phos-
phopantetheinyl transferase (PPTase) Sfp15,16 with BODIPY-
CoA14 to produce a fluorescent band during SDS-PAGE
analysis (Figure S5, Supporting Information).
DdaC Is an Fe(II)/r-KG-Dependent Epoxidase. When DdaC
was added to reaction mixtures containing NꢀFmmDAP-S-DdaD
and R-KG, a +16 Da mass shift in the intact mass of both the
T domain active site tryptic peptide and small molecule Ppant
ejection ion (to yield an ion at m/z 460.2) was observed (Figure
4; Table S3, Supporting Information). This mass shift was
observed following two types of enzymatic incubations: (1)
reaction mixtures containing DdaC that had been incubated
anaerobically with Fe(NH4)2(SO4)2 and R-KG and then desalted
(Figure 4) and (2) reaction mixtures containing aerobically
purified DdaC to which excess Fe(NH4)2(SO4)2 was added
(Figure S11, Supporting Information). No qualitative differences
in the activities of the two enzyme preparations were observed,
so the latter method was used for the remaining experiments.
No +16 Da mass shift was observed in DdaC incubations that
lacked R-KG (Figure S12, Supporting Information). In contrast
to the activity of DdaC on NꢀFmmDAP-S-DdaD, we have
observed no evidence for DdaC oxidation of free NꢀFmmDAP
by LC-MS or HPLC assay.
Next we turned to FTMS and the Ppant ejection assay20,21
(Scheme S1, Supporting Information) to further characterize
intermediates tethered to DdaD. The experiments were carried
out in one of two fashions. For some analyses, enzymatic
incubations were subjected to trypsin digestion, and tryptic
peptides were separated by RPLC coupled directly to a hybrid
linear ion trap FTMS system (ThermoFisher Scientific LTQ-
FT), allowing determination of the masses of intact peptides,
identification of the DdaD active site peptide (containing the
site of serine (Ser) phosphopantetheinylation (PPTation)), and
observation of Ppant ejection products. In other experiments,
undigested reactions were subjected to RPLC-MS, and Ppant
ejection was observed directly from intact DdaD.
During LC-MS analysis of a tryptic digest of apo-DdaD
incubated with Sfp and CoA, we observed a thiolation (T)
To confirm that the oxidation catalyzed by DdaC was indeed
an epoxidation, we compared the MSn fragmentation patterns
of the Ppant ejection ion generated from HS-DdaD loaded with
synthetic Nꢀ-trans-EpSmDAP (both diastereomers) and the
Ppant ejection ion generated from NꢀFmmDAP-S-DdaD incu-
bated with DdaC. Fragment ions for MSn analysis were selected
that reported a +16 Da shift when compared to similar analyses
(20) Dorrestein, P. C.; Bumpus, S. B.; Calderone, C. T.; Garneau-Tsodikova,
S.; Aron, Z. D.; Straight, P. D.; Kolter, R.; Walsh, C. T.; Kelleher,
N. L. Biochemistry 2006, 45, 12756–12766.
(21) Bumpus, S. B.; Kelleher, N. L. Curr. Opin. Chem. Biol. 2008, 12,
475–482.
9
J. AM. CHEM. SOC. VOL. 132, NO. 44, 2010 15777

A R T I C L E S
Hollenhorst et al.
Figure 3. Apo-DdaD is converted to holo-DdaD (HS-DdaD) in the presence of Sfp and CoA. In the presence of ATP and NꢀFmmDAP, HS-DdaD is loaded
with NꢀFmmDAP to form NꢀFmmDAP-S-DdaD. (A) In the presence of Sfp but the absence of CoA, only the active site peptide from apo-DdaD (green) is
observed. (B) In the presence of Sfp and CoA, the active site peptide from HS-DdaD (red) is observed. This peptide shows a mass shift of +340.09 Da from
the apo-DdaD active site peptide, the exact mass shift expected for PPTation. (C) The small molecule Ppant ejection ion (red) is observed from the HS-DdaD
active site peptide when the peptide is subjected to MS/MS by NSD. (D) Both predicted Ppant ejection peptide marker ions are observed when the HS-DdaD
active site peptide is subjected to MS/MS using CID. (E) In the presence of ATP but the absence of NꢀFmmDAP, only the HS-DdaD active site peptide (red)
is observed. (F) In the presence of both ATP and NꢀFmmDAP, both the HS-DdaD (red) and NꢀFmmDAP-S-DdaD (blue) active site peptides are observed.
(G) When the HS-DdaD and NꢀFmmDAP-S-DdaD active site peptides are subjected to MS/MS using NSD, both expected small molecule Ppant ejection
ions are observed. The HS-DdaD Ppant ejection ion is shown in red, and the NꢀFmmDAP-S-DdaD Ppant ejection is shown in blue. For the predicted
structures of the Ppant ejection ions, see Figure S6 (Supporting Information).
conducted on the Ppant ejection ion from NꢀFmmDAP-S-DdaD.
In all cases, the fragmentation patterns produced were the same
for all epoxide-containing species, confirming the DdaC-
catalyzed oxidation as an epoxidation (Figure S13, Supporting
Information).
To determine the origin of the putative incorporated oxygen,
DdaC incubations were carried out either in H218O or under an
18O2 atmosphere. When the reaction was conducted in H218O
and then analyzed by trypsin digest followed by LC-MS, a
+16 Da mass shift was observed in the DdaD tryptic active
site peptide and the corresponding Ppant ejection ion. If the
oxygen atom were derived from water, a +18 Da mass shift
would be observed (Figure S14, Supporting Information). In
contrast, incubations under 18O2 followed by LC-MS and Ppant
ejection analysis of intact DdaD resulted in a +18 Da (as
opposed to a +16 Da mass shift in a 16O2 atmosphere) mass
shift in the observed NꢀEpSmDAP-S-DdaD Ppant ejection ion,
indicating that DdaC uses O2 as a cosubstrate (Figure 5; Table
S3, Supporting Information).
Discussion
Figure 4. In the presence of both R-KG and DdaC, NꢀFmmDAP-S-DdaD
The dapdiamides comprise a family of acylated dipeptide
is converted to NꢀEpSmDAP-S-DdaD. (A) In the presence of R-KG but
natural antibiotics that likely inhibit the glucosamine-6-
phosphate synthase enzyme of susceptible organisms and
consequently disrupt cell wall assembly and integrity.1 The
putative target has an active site Cys that can be covalently
captured by the warheads of the dapdiamides and related
scaffolds.2 Most commonly, the electrophilic warhead is the
enamide moiety provided by the fumaramoyl group of this
antibiotic class (e.g., dapdiamides A-D), which can be a
Michael acceptor for the Cys thiolate. An alternative elec-
trophile is found in the R-epoxysuccinamoyl-containing
dapdiamide E and in the corresponding epoxysuccinamoyl
the absence of DdaC, only the active site peptide from NꢀFmmDAP-S-
DdaD (blue) is observed. (B) In the presence of both R-KG and DdaC
(prepared anaerobically), NꢀFmmDAP-S-DdaD is converted to NꢀEpSmDAP-
S-DdaD and the active site peptide from NꢀEpSmDAP-S-DdaD (orange) is
observed. The theoretical mass shift of this transformation is 15.995 Da.
(C) In the presence of R-KG and the absence of DdaC, only the small
molecule Ppant ejection ion from NꢀFmmDAP-S-DdaD (blue) is observed
when the peptides are subjected to MS/MS using NSD. (D) In the presence
of both R-KG and DdaC (prepared anaerobically), the Ppant ejection ions
from both NꢀFmmDAP-S-DdaD and NꢀEpSmDAP-S-DdaD (orange) are
observed when the peptides are subjected to MS/MS using NSD. For the
predicted structures of the Ppant ejection ions, see Figure S6 (Supporting
Information).
9
15778 J. AM. CHEM. SOC. VOL. 132, NO. 44, 2010

Epoxidation in Dapdiamide Biosynthesis
A R T I C L E S
the two ATP-cleaving amide-forming ligases that are responsible
for assembling the Fmm-dipeptide scaffold. DdaG uses ATP
to activate fumarate to fumaroyl-AMP on the way to making
NꢀFmDAP. DdaF then makes the second amide bond, but only
after the acid of FmDAP has been converted enzymatically to
the amide in FmmDAP. DdaF is a member of the ATP grasp
family,22 and as such cleaves ATP to ADP and Pi, presumably
making the FmmDAP-phosphate mixed anhydride as an acti-
vated intermediate. To date only dapdiamide enzymes that form
and utilize Nꢀ-acyl-DAP species have been characterized; the
enzyme creating the NRFmmDAP linkage has not yet been
identified.
In this study, we assayed both synthetic Nꢀ-trans-EpSmDAP
diastereomers and validated that they are substrates for DdaF
in the presence of Val and ATP to produce the NꢀEpSmDAP-
Val dipeptide. NꢀEpSmDAP-Val is the natural product (of as
yet unassigned epoxide stereochemistry) recently identified in
P. agglomerans strains (48b/90, 39b/90, and C9-1) as well as a
strain of Serratia plymuthica.5,6 We observed selectivity of DdaF
for NꢀRREpSmDAP over the (S,S) diastereomer, suggesting that
the dapdiamide pathway produces a natural product with (R,R)-
epoxide stereochemistry. The precedence of (R,R)-epoxide
stereochemistry in the related natural product Sch37137 is in
accord with this hypothesis.8
Sequence analysis of the Dda gene cluster suggested that, in
addition to DdaG and DdaF, one additional ORF should be
capable of using ATP to activate an acid cosubstrate, namely,
DdaD. This protein is predicted to be a member of a third family
of ATP-cleaving enzymes and contains two domains which
comprise a minimal NRPS module. The first domain of
approximately 50 kDa is predicted to be an A domain, while
the second of 10 kDa should be a T domain that can be post-
translationally primed with a Ppant group. The adjacent ORF
DdaE, encoding a predicted thioesterase, is the only other NRPS-
related ORF in the dapdiamide biosynthetic gene cluster,
suggesting the adenylation-thiolation (A-T) didomain DdaD
acts as a stand-alone module and not as part of a classical NRPS
assembly line.
Figure 5. In an atmosphere of 18O2, 18O is incorporated into the substrate
loaded onto DdaD and NꢀFmmDAP is converted to Nꢀ-trans-EpSmDAP.
(A) Under normal reaction conditions (an atmosphere of 16O2), in the
presence of R-KG and the absence of DdaC, the only Ppant ejection ion
observed (blue) during Ppant ejection analysis (using NSD) of NꢀFmmDAP-
S-DdaD corresponds to the unoxidized starting substrate. (B) Under normal
reaction conditions (an atmosphere of 16O2), in the presence of both R-KG
and DdaC, Ppant ejection ions corresponding to both NꢀFmmDAP-S-DdaD
and NꢀEpSmDAP-S-DdaD (orange) are observed. (C) Under an atmosphere
of 18O2, in the presence of R-KG but the absence of DdaC, the only Ppant
ejection ion observed corresponds to NꢀFmmDAP-S-DdaD. (D) Under an
atmosphere of 18O2, in the presence of both R-KG and DdaC, Ppant ejection
ions are observed for both NꢀFmmDAP-S-DdaD and NꢀEpSmDAP-S-DdaD.
A +2 Da mass shift (from the NꢀEpSmDAP-S-DdaD Ppant ejection ion in
(B)) is observed in the Ppant ejection ion corresponding to NꢀEpSmDAP-
S-DdaD (purple), indicating that the oxygen in the epoxide originates from
molecular oxygen. For the predicted structures of the Ppant ejection ions
see Figure S6, Supporting Information.
We have shown in several other contexts that stand-alone
A-T didomains (or isolated T domains) are used in bacterial
metabolism to sequester some fraction of an amino acid pool,
tethered as the aminoacyl-S-pantetheinyl T domain thioester.
The tethered aminoacyl-S-T domain is then subjected to covalent
modification, which is often oxidative. Thus, in the biosynthesis
of the vancomycin class of glycopeptides23,24 and the nikko-
mycin25 and aminocoumarin26 classes of antibiotics, Cꢀ hy-
droxylation of the sequestered aminoacyl thioester moiety is
effected by either heme iron or nonheme iron oxygenases. In
the biogenesis of syringomycin, chlorination occurs at C4 of a
tethered threonyl moiety,27 while in the assembly of the
jasmonate phytohormone mimic coronatine, cryptic chlorination
occurs at Cγ of the allo-Ile moiety prior to subsequent ring
closure to the cyclopropane.28,29
ꢀ regioisomer NꢀEpSmDAP-Val (Figure 1C), in which the
olefin of the fumaramoyl moiety is replaced with an epoxide
that is proposed to be opened by the active site Cys residue.
The presumption has been that the fumar(am)oyl group gets
epoxidized to the epoxysuccin(am)oyl group to create this
second potential electrophile; the timing and the identity of
the catalysts responsible for this conversion are the subject
of this study.
Regiochemical variation is observed among the dapdiamides
with regard to which amino group of 2(R),3(ꢀ)-DAP the
fumaramoyl/epoxysuccinamoyl moieties are attached in amide
linkage. Dapdiamides A-C have an NꢀFmmDAP attachment,
while dapdiamide D has an NRFmmDAP linkage. Dapdiamide
E, with an epoxysuccinamoyl group in R amide linkage, could
in principle arise from epoxidation of dapdiamide D or its likely
precursor amino acid NRFm(m)DAP. The natural product
NꢀEpSmDAP-Val could arise from comparable epoxidation of
dapdiamide A or NꢀFm(m)DAP. In this work we demonstrate
that it is NꢀFmmDAP that is the substrate for T domain loading
and then epoxidation.
(22) Galperin, M. Y.; Koonin, E. V. Protein Sci. 1997, 6, 2639–2643.
(23) Chen, H.; Thomas, M. G.; O’Connor, S. E.; Hubbard, B. K.; Burkart,
M. D.; Walsh, C. T. Biochemistry 2001, 40, 11651–11659.
(24) Hubbard, B. K.; Walsh, C. T. Angew. Chem., Int. Ed. 2003, 42, 730–
765.
(25) Chen, H.; Hubbard, B. K.; O’Connor, S. E.; Walsh, C. T. Chem. Biol.
2002, 9, 103–112.
(26) Chen, H.; Walsh, C. T. Chem. Biol. 2001, 8, 301–312.
(27) Vaillancourt, F. H.; Yin, J.; Walsh, C. T. Proc. Natl. Acad. Sci. U.S.A.
2005, 102, 10111–10116.
In our prior study of the biosynthetic pathway for this
antibiotic family, we demonstrated that DdaG and DdaF are
9
J. AM. CHEM. SOC. VOL. 132, NO. 44, 2010 15779

A R T I C L E S
Hollenhorst et al.
With such precedents, we sought an analogous role for
modification of an aminoacyl thioester covalently attached to
DdaD. The adjacent ORF DdaC has homology to the Fe(II)/
R-KG-dependent dioxygenase family of enzymes, which typi-
cally catalyze O2-dependent substrate hydroxylations.12 Ex-
amples of members of this family include the syringomycin
biosynthetic enzyme SyrP30 and the kutzneride pathway en-
zymes KtzO and P,31 which carry out ꢀ-hydroxylations of T
domain-tethered aspartate and glutamate, respectively. In ad-
dition to hydroxylations, members of this family have been
shown to carry out a range of other oxidative transformations.12
Evidence from bioconversion and cell extract studies has
implicated Fe(II)/R-KG enzymes in epoxidation reactions,32,33
but to our knowledge no in vitro characterization of a purified
epoxidase in this class has been reported previously.
In the context of the known dapdiamide family members
(Figure 1A) it seemed likely that DdaC could be an epoxidase
that acts on the fumaroyl/fumaramoyl moiety of an intermediate
tethered in thioester linkage to the T domain of DdaD. In
addition, DdaE is a predicted thioesterase; thus, the tandem
action of DdaD, C, and E could be a branch pathway for
selection and activation of an olefin-containing pathway inter-
mediate, epoxidation, and then hydrolysis to produce an
epoxysuccin(am)oyl building block for condensation with
another monomer via DdaG and/or DdaF. (We have not been
able to heterologously express DdaE in a soluble form in E.
coli to establish such a thioesterase role.)
Validation of the proposed roles for DdaD and DdaC started
with determination of the selectivity of the A domain of DdaD.
Using the classical ATP-[32P]PPi exchange assay, diagnostic
for reversible formation of tightly held (amino)acyl-AMPs in
enzyme active sites, DdaD showed clear preference for
NꢀFmmDAP. The Km value for DdaD with respect to
NꢀFmmDAP was found to be 420 µM, comparable to Km values
reported for other NRPS A domains.28,34,35 These results
provided a key early insight: DdaD is indeed selecting an olefin-
containing pathway intermediate for activation as the AMP
mixed anhydride. This was strongly suggestive that the fuma-
ramoyl moiety of thioester-tethered FmmDAP would be the
species epoxidized.
To validate the second step of A-T didomain function, the
predicted covalent loading of NꢀFmmDAP-AMP onto the Ppant
arm of the T domain of DdaD, we turned to mass spectrometry.
We found that apo-DdaD could be post-translationally converted
to the Ppant-containing holo-DdaD by action of purified Sfp.
Incubation of holo-DdaD with ATP and NꢀFmmDAP allowed
detection of the NꢀFmmDAP-S-Ppant adduct in the T domain
by peptide mass analysis and by the release of the NꢀFmmDAP-
S-Ppant thioester fragment ion. Thus, the second step of A-T
didomain function, the covalent tethering of the substrate
activated by the A domain, was operant.
When DdaC was incubated with the covalent NꢀFmmDAP-
S-DdaD enzyme intermediate, a mass increase of +16 Da was
observed for both the T domain active site tryptic peptide
containing the tethered acyl-DAP thioester and the ejected Ppant
ion. We found that, as anticipated for a member of the Fe(II)/
R-KG family, the activity of DdaC is dependent on R-KG.
Additionally, incubation under 18O2(g) resulted in a +18 Da
mass shift, demonstrating that DdaC uses molecular oxygen as
a cosubstrate.
The ejected pantetheinyl fragment from DdaC/D experiments
had the M + 16 Da mass increase anticipated for the epoxide
product. However, it was formally possible that the introduction
of one oxygen atom into the FmmDAP moiety arose not by
epoxidation of the double bond but by C- or N-hydroxylation
of the DAP residue. MSn fragmentation of Ppant ejection ions
from both HS-DdaD loaded with authentic Nꢀ-trans-EpSmDAP
and from NꢀFmmDAP-S-DdaD incubated with DdaC resulted
in the same fragmentation pattern, suggesting that DdaC indeed
acts as an epoxidase.
Our studies of DdaC have generated a number of questions
to be answered in future investigations. We have not
attempted to determine single-turnover kinetics of the enzyme
because of the difficulty of quantifying its substrate, the
covalent N-acylaminoacyl thioester adduct of DdaD. We have
also been unable to obtain sufficient NꢀEpSmDAP from
DdaC/D incubations to directly determine the stereochemistry
of the epoxide carbons in the product, nor have we yet
evaluated the epoxidation mechanism. In analogy to proposed
mechanisms for Fe(II)/R-KG hydroxylases, an Fe(IV)-oxo
intermediate is the likely oxygen transfer species. However,
whether C-O bond formations are stepwise and ionic or
radical, as suggested in Scheme S2 (Supporting Information),
is yet to be probed.
Additionally, the question arises of why P. agglomerans
makes both the enamide electrophile (fumaramoyl) and the
epoxide electrophile (epoxysuccinamoyl) as parallel N-acyl
warheads in this antibiotic family. Two future studies will
compare the epoxysuccinamoyl versus the fumaramoyl
groups. First, minimum inhibitory concentration (MIC)
determinations of the Nꢀ molecules dapdiamide A and
NꢀEpSmDAP-Val will test for any differences in uptake by
susceptible bacteria and fungi. Once taken up by the
oligopeptide permease systems, intracellular proteases are
thought to liberate the Nꢀ-acyl-DAPs as the proximal
inactivators for glucosamine synthase. Thus, it will be useful
to compare FmmDAP and EpSmDAP side by side against
the target enzyme to determine inactivation efficiencies. It
is possible that the epoxide warhead is more selective than
the enamide: the epoxide may require acid catalysis in the
enzyme active site for covalent capture, whereas Michael
addition to the fumaramoyl moiety may not. In that context
a proteomics36 study to evaluate how many proteins in a
susceptible cell are targeted covalently would offer a global
comparison of “off-target” labeling by the two types of
electrophilic N-acyl warheads.
(28) Couch, R.; O’Connor, S. E.; Seidle, H.; Walsh, C. T.; Parry, R. J.
Bacteriol. 2004, 186, 35–42.
(29) Vaillancourt, F. H.; Yeh, E.; Vosburg, D. A.; O’Connor, S. E.; Walsh,
C. T. Nature 2005, 436, 1191–1194.
(30) Singh, G. M.; Fortin, P.; Koglin, A.; Walsh, C. T. Biochemistry 2008,
47, 11310–11320.
(31) Strieker, M.; Nolan, E. M.; Walsh, C. T.; Marahiel, M. A. J. Am.
Chem. Soc. 2009, 131, 13523–13530.
(32) Watanabe, M.; Sumida, N.; Murakami, S.; Anzai, H.; Thompson, C. J.;
Tateno, Y.; Murakami, T. Appl. EnViron. Microbiol. 1999, 65, 1036–
1044.
Acknowledgment. We thank Emily Balskus, Christopher Neu-
mann, Elizabeth Sattely, and Albert Bowers for helpful discussions.
We thank John Heemstra for providing synthetic BODIPY-CoA,
(33) Hashimoto, T.; Matsuda, J.; Yamada, Y. FEBS Lett. 1993, 329, 35–
39.
(34) Mootz, H. D.; Marahiel, M. A. J. Bacteriol. 1997, 179, 6843–6850.
(35) Ehmann, D. E.; Shaw-Reid, C. A.; Losey, H. C.; Walsh, C. T. Proc.
Natl. Acad. Sci. U.S.A. 2000, 97, 2509–2514.
(36) Han, X.; Aslanian, A.; Yates, J. R., III. Curr. Opin. Chem. Biol. 2008,
12, 483–490.
9
15780 J. AM. CHEM. SOC. VOL. 132, NO. 44, 2010

Epoxidation in Dapdiamide Biosynthesis
A R T I C L E S
Elizabeth Sattely for providing Sfp, and Jessica Dawlaty for
providing synthetic dapdiamide A and the pUC19 A10A plasmid.
This work was supported in part by NIH Grant GM 20011 (C.T.W.),
NIH Medical Scientist Training Program GM 07753 (M.A.H.), NIH
Grant GM 067725-08 (N.L.K.), NSF Grant MCB-642058 (J.M.B.),
and a fellowship from the American Chemical Society Division of
Analytical Chemistry (S.B.B.).
Supporting Information Available: Additional materials and
methods, Tables S1-S5, Figures S1-S14, and Schemes S1-S2.
This material is available free of charge via the Internet at http://
pubs.acs.org.
JA1072367
9
J. AM. CHEM. SOC. VOL. 132, NO. 44, 2010 15781