Biosynthesis of (-)-(1S,2R)-allocoronamic acyl thioester by an Fe II-dependent halogenase and a cyclopropane-forming flavoprotein

Article abstract of DOI:10.1021/ja8064667

The biosynthetic gene cluster for the kutzneride family of hexapeptidolactones includes the four-gene cassette ktzABCD postulated to generate a nonproteinogenic amino acid. Encoded by this cassette are the nonheme Fe^II-dependent halogenase KtzD

Full text of DOI:10.1021/ja8064667

Published on Web 10/02/2008
Biosynthesis of (-)-(1S,2R)-Allocoronamic Acyl Thioester by an
FeII-Dependent Halogenase and a Cyclopropane-Forming Flavoprotein
Christopher S. Neumann and Christopher T. Walsh*
Department of Biological Chemistry and Molecular Pharmacology, HarVard Medical School, Boston,
Massachusetts, 02115
Received August 14, 2008; E-mail: Christopher_Walsh@hms.harvard.edu
Nonribosomal peptides constitute a diverse class of secondary
metabolites with important biological activities.1 In addition to the
twenty proteinogenic amino acids, nonribosomal peptide synthetases
make use of a large number of modified amino acid and hydroxy
acid building blocks.2 Understanding the chemical pathways and
mechanisms used in the biosyntheses of these monomers is a first
step toward expanding the pool of new molecules available through
metabolic engineering.
Toward this end, we recently used halogenase-directed probes
to clone the gene cluster3 responsible for the biosynthesis of a series
of hexadepsipeptides, kutznerides, isolated from the soil actino-
mycete kutzneria4,5 (Figure S1). These molecules contain a number
of unusual monomers whose biosyntheses have not been described.
Bioinformatic analysis of the gene cluster identified three potential
halogenases. KtzQ and KtzR are shown in separate work to be
flavin-dependent tryptophan halogenases which can generate the
6,7-dichlorotryptophan residue postulated in kutzneride biosynthe-
sis.6 KtzD was predicted to be an FeII-dependent halogenase capable
of chlorinating an unactivated carbon center.
The four-protein cassette KtzABCD could potentially function
to generate a novel amino acid via a cryptic chlorination pathway.
KtzC and KtzB were assigned roles as carrier protein and
adenylation protein, respectively. KtzA is a flavoprotein homologous
to members of the acyl-CoA dehydrogenase (ACAD) family of
enzymes. We initially hypothesized that these four proteins produce
chlorinated intermediate (2) which is dehydrochlorinated by KtzA (3). (B)
Figure 1. (A) HPLC and MS analysis of cleaved reaction products as
isoindole derivatives. Treatment of L-Ile-S-KtzC (1) with KtzD yields a
the nonproteinogenic amino acid 2-(1-methylcyclopropyl)glycine
(MecPG),3,7 a component of all kutzernide molecules reported to
date. Herein, we report the biochemical characterization of Ktz-
ABCD and demonstrate that they instead function to produce an
alternative cyclopropyl-containing amino acid, (1S,2R)-allocoro-
namic acid (alloCMA), tethered to KtzC. To our knowledge, this
is the first identification of alloCMA in a natural source.
To study the role of the KtzABCD cassette, the individual genes
were cloned into E. coli expression vectors as His-tag fusions.
Overexpression and Ni-NTA purification provided soluble protein
for each construct. The carrier protein KtzC was isolated in apo
form and phosphopantetheinylated via incubation with coenzyme
A and Sfp. KtzA was isolated with FAD bound at 50% occupancy.
After initial purification of the halogenase KtzD, the His tag was
proteolytically cleaved and the enzyme purified by gel filtration
and reconstituted anaerobically with FeII and R-ketoglutarate to
obtain the active form of the protein.
The biosynthetic pathway for allocoronamic acid tethered in thioester linkage
to KtzC. A detailed mechanism is presented in Scheme S1.
product (Figure 1A). MS analysis on the released isoindole
derivative shows this product results from a single chlorination
event. Treatment of L-allo-Ile-S-KtzC with KtzD does not lead to
the formation of a new product (Figure S3), illustrating the substrate
specificity of the halogenase.
Exposure of the chlorinated intermediate to KtzA again leads to
complete conversion to a new product. MS analysis shows the
released amino acid product to have a m/z ratio consistent with
dehydrochlorination of the intermediate and net two-electron
oxidation of the starting isoleucine. Supplementing the reaction with
excess FAD had no effect on product formation (data not shown).
To confirm the structure of the final product, the isoindole derivative
was purified by HPLC and analyzed by 1H- and COSY-NMR.
Additionally, 13C6-Ile was used as the starting material, and the
isotope-enriched product analyzed by HMQC and 1H NMR
spectroscopy. The spectral data support the atom connectivity of
alloCMA (Figures S7, S8, Table S1).
In accordance with its previously described adenylating activity,3
KtzB efficiently activates and loads both L-Ile and L-allo-Ile to holo-
KtzC as the phosphopantetheinyl thioesters. The protein-tethered
amino acids can be identified by enzymatic hydrolysis with the
type II thioesterase TycF8 and conversion to readily detectable
isoindole derivatives using established procedures.9 Incubation of
L-Ile-S-KtzC with KtzD for 1 h results in complete consumption
of starting material and production of a new aminoacyl thioester
The stereochemistry of the product was deduced from two
observations. First, the observed product fails to coelute with an
authentic sample of coronamic acid under the typical HPLC
conditions (Figure S9). Second, the γ-CH2 protons show 1H NMR
resonances at 1.47 and -0.14 ppm. The unusual upfield resonance
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14022 J. AM. CHEM. SOC. 2008, 130, 14022–14023
10.1021/ja8064667 CCC: $40.75
2008 American Chemical Society

C O M M U N I C A T I O N S
to the FAD coenzyme has previously been observed for the
flavoprotein D-alanine oxidase when presented with a non-natural
substrate.13 In that case, ꢀ-chloroalanine can be partitioned between
R,ꢀ-dehydrochlorination to dehydroalanine and CR oxidation to
ꢀ-chloro-iminopyruvate. In KtzA catalysis, the net removal of HR
and Clγ appears to be the physiological reaction. We have previously
reported a related four protein pathway in coronamic acid biosyn-
thesis where L-allo-Ile is comparably converted to (1S,2S)-coro-
namic acid via the same cryptic γ-chlorination strategy, but in that
case the cyclization catalyst is the Zn-dependent enzyme CmaC.9,14
KtzA and CmaC thus represent two distinct enzymatic strategies
for generation of the thioester enolate needed to initiate cyclopro-
pane formation.
In conclusion, we have characterized the activities of the
KtzABCD proteins and identified the novel metabolite alloCMA
as the protein-tethered product of these enzymes. Deuterium-
labeling studies confirm the regioselective chlorination of L-Ile-S-
KtzC at the γ-CH3 position by KtzD and support the unusual role
of the flavoprotein KtzA as the catalytic base which induces
cyclization without redox participation of bound FAD. This cassette
provides a biosynthetic route to a novel amino acid monomer that
is a stereochemical complement to the previously described
coronamic acid. At the same time, the results suggest future
directions for research on kutzneria. Identifying additional nonri-
bosomal peptides which may contain alloCMA and locating the
gene cassette which provides for MecPG biosynthesis are now key
avenues for metabolomic and genomic approaches.
Figure 2. Treatment of deuterated Ile-S-KtzC isotopologues with KtzD
indicates regioselective chlorination at the γ-CH3 position.
is best explained by strong shielding of the proton as a result of its
location directly above the aromatic ring of the isoindole group.
For this to occur on the rigid cyclopropyl skeleton, the ethyl side
chain and isoindole group must be in a cis arrangement. On the
basis of the observed alloCMA structure, we propose a biosynthetic
scheme which involves γ-CH3 chlorination of L-Ile-S-KtzC followed
by KtzA-mediated R,γ cyclization to alloCMA-S-KtzC via an
intermediate CR-carbanion (Figure 1B).
To confirm the regiochemistry of γ-chlorination by KtzD, we
synthesized substrates deuterated at the γ-CH3 position (Ile-d3) or
at the ꢀ-CH,γ-CH2, and δ-CH3 positions (Ile-d6). Treatment of Ile-
d6-S-KtzC with KtzD produces a chlorinated product that retains
all deuterium labels. Identical treatment of Ile-d3-S-KtzC produces
a chlorinated product which retains only two deuterium labels
(Figure 2). Additionally, analysis of the reaction at partial conver-
sion shows that the rate of chlorination of Ile-d3 is retarded relative
to Ile and Ile-d6 (Figure S4). This kinetic isotope effect for H/D
abstraction is consistent with the isotope effect observed in stopped
flow studies of the related halogenase CytC3.10
Acknowledgment. We thank Dr. Danica Galonic Fujimori for
technical assistance and helpful discussions. We thank Dr. Alex
Koglin and Dr. Dominique Frueh for assistance in obtaining HMQC
spectra. This work was supported by NIH grant GM20011 to
C.T.W.
Supporting Information Available: Supplemental figures, experi-
mental procedures and spectral data. This material is available free of
charge via the Internet at http://pubs.acs.org.
References
Cyclization of 2 to alloCMA-S-KtzC is mediated by the ACAD-
like flavoprotein KtzA. ACADs catalyze dehydrogenation of acyl/
aminoacyl CoA thioesters by abstraction of the acidic R-proton and
transfer of a ꢀ-hydride to the FAD cofactor to yield the R,ꢀ-
unsaturated thioester and FADH2.11 In contrast, KtzA needs only
abstract the R-proton of 2 to generate a stabilized thioester enolate
which can collapse by intramolecular attack at Cγ to form the
cyclopropane. Loss of Cl- constitutes the two-electron oxidation
of substrate and avoids formation of the FADH2 oxidation state.12
To verify that KtzA can catalyze reversible R-proton abstraction
as the initial event in cyclization, unlabeled Ile-S-KtzC was
incubated with KtzA in the presence of D2O. Recovery of the amino
acid and analysis by HPLC and MS showed incorporation of a
single deuterium atom (Figures S5, S6). The yield is similar to that
for untreated Ile-S-KtzC indicating that there is no significant loss
of amino acid through hydrolysis of a dehydrogenated intermediate.
Attempts to generate apo-KtzA by treatment of Ni-bound protein
with 2 M KBr and 2 M urea gave negligible yields of protein; thus
it is possible the flavin cofactor is needed to maintain the tertiary
structure of KtzA.
(1) Felnagle, E. A.; Jackson, E. E.; Chan, Y. A.; Podevels, A. M.; Berti, A. D.;
McMahon, M. D.; Thomas, M. G. Mol. Pharm. 2008, 5, 191–211.
(2) Do¨hren, H. v. In Biochemistry of Peptide Antibiotics; Do¨hren, H. v.,
Kleinkauf, H., Eds.; de Gruyter: Berlin, NY, 1990; p 411-506.
(3) Fujimori, D. G.; Hrvatin, S.; Neumann, C. S.; Strieker, M.; Marahiel, M. A.;
Walsh, C. T. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 16498–16503.
(4) Broberg, A.; Menkis, A.; Vasiliauskas, R. J. Nat. Prod. 2006, 69, 97–102.
(5) Pohanka, A.; Menkis, A.; Levenfors, J.; Broberg, A. J. Nat. Prod. 2006,
69, 1776–1781.
(6) Heemstra, J. R.; Walsh, C. T. J. Am. Chem. Soc. 2008, 130, 14024–14025.
(7) Neumann, C. S.; Fujimori, D. G.; Walsh, C. T. Chem. Biol. 2008, 15, 99–
109.
(8) Yeh, E.; Kohli, R. M.; Bruner, S. D.; Walsh, C. T. Chembiochem 2004, 5,
1290–3.
(9) Vaillancourt, F. H.; Yeh, E.; Vosburg, D. A.; O’Connor, S. E.; Walsh,
C. T. Nature 2005, 436, 1191–1194.
(10) Fujimori, D. G.; Barr, E. W.; Walsh, C. T.; Bollinger, J. M.; Krebs, C.
Nat. Chem. Biol. 2007, 3, 113–116.
(11) (a) Ghisla, S.; Thorpe, C. Eur. J. Biochem. 2004, 271, 494–508. (b)
Mansoorabadi, S. O.; Thibodeaux, C. J.; Liu, H. J. Org. Chem. 2007, 72,
6329–6342.
(12) While not common, redox inactive flavoproteins are known. For examples,
see ref 11b and: Bornemann, S. Nat. Prod. Rep. 2002, 19, 761–772.
(13) Walsh, C. T.; Schonbrunn, A.; Abeles, R. H. J. Biol. Chem. 1971, 246,
6855–6866.
(14) Kelly, W. L.; Boyne, M. T.; Yeh, E.; Vosburg, D. A.; Galonic, D. P.;
Kelleher, N. L.; Walsh, C. T. Biochemistry 2007, 46, 359–368.
The rerouting of a carbanion intermediate in a flavoprotein active
site to intramolecular elimination rather than two-electron transfer
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