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
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
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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.
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