C O M M U N I C A T I O N S
porting Information). This increase is consistent with an R,â CdC
bond formation in an acyl-CoA thioester.9 In addition, MS/MS
analysis confirmed that the dehydration product was 6-CoA, and
we did not pursue the further clarification of the CdC configuration.
To determine regiochemistry of the double bond in the decarboxy-
lation product, we synthesized 3-methyl-3-butenoyl-CoA and used
commercially available 7-CoA (Sigma) as authentic standards.
HPLC co-injection showed that the decarboxylation product is
7-CoA (see Supporting Information).
Finally, we investigated the substrate preference of ECH1 by
comparing the conversion ratio of (R,S)-HMG-CoA and (S)-HMG-
CoA, which was generated by HMG-CoA reductase.10 On the basis
of HPLC traces, the conversion ratio of (S)-HMG-CoA is 2-fold
higher than that of (R,S)-HMG-CoA (see Supporting Information),
which indicates that (S)-HMG is the natural ECH1 substrate.11
In summary, CurE/CurF ECH1-ECH2 polypeptides from the
curacin A biosynthetic pathway were functionally identified as a
mechanistically diverse enzyme pair. We demonstrated that CurE
ECH1 catalyzes dehydration of (S)-HMG-ACP 5 to form 3-meth-
ylglutaconyl-ACP 6, and CurF ECH2 catalyzes decarboxylation of
6 to generate 3-methylcrotonyl-ACP 7, the presumed precursor for
cyclopropyl-ACP 8 formation in curacin A. The detailed steps
leading from 3-methylcrotonyl-ACP to the cyclopropane ring are
the subject of ongoing studies in our laboratory. It is noteworthy
that, to date, only two members of the crotonase enzyme super-
family, methylmalonyl CoA decarboxylase (YgfG)12 and CarB,13
were reported to catalyze loss of carbon dioxide. Thus, identification
of the reaction catalyzed by CurF ECH2 provides a new example
of this novel biotin-independent decarboxylase in secondary
metabolism.
Figure 2. ECH1 and ECH2 assays for the substrates in CoA and ACP forms.
(A) FTICR spectra, 50 µM (R,S)-HMG-ACP, 2 µM ECH1, ECH2, or both,
at 37 °C for 3 h. Experimental most abundant mass: 5-ACP, 11325.8;
6-ACP, 11307.8; 7-ACP, 11264.8. (B) UV 275 nm traces of (a) standards:
5-CoA and 7-CoA (MC-CoA), 0.5 mM (R,S)-5-CoA treated with (b)
2 µM ECH1, (c) 2 µM ECH2, (d) 2 µM ECH1 and ECH2 at 37 °C for 3 h.
The CoA peak shoulders and the following minor peaks are possibly due
to CoA aggregation.
Acknowledgment. We thank Professor Christopher Walsh, Dr.
Christopher Calderone, and Dr. Sabine Gru¨schow for helpful
discussions, Dr. Christopher Calderone for the 3-methyl-3-butenoyl-
CoA standard, Professor Victor Rodwell for HMG-CoA reductase,
and Dr. Kate Noon, Biomedical Mass Spectrometry Facility at the
University of Michigan, for technical assistance. This work was
supported by NIH Grant CA108874 to D.H.S. H.L. is supported
by a Searle Scholar award to K.H.
via in vitro phosphopantetheinylation through in situ incubation
with (R,S)-HMG-CoA (500 µM) and Sfp (4 µM).8 The HMG-holo-
CurB was dialyzed against the ECH assay buffer to adjust pH and
remove excess HMG-CoA.
The in vitro activities of ECH1 and ECH2 were investigated by
incubating ECH1, ECH2, or both (2 µM each), with (R,S)-HMG-
holo-CurB (50 µM) in a series of buffers at 37 °C. ESI-FTMS
(Apex-Q instrument, Bruker Daltonics) was applied to detect mass
change of the acyl group covalently linked to the holo-CurB. We
found that, in the presence of ECH2 alone (Figure 2A, c), no new
reaction products were observed. In contrast, in the presence of
ECH1, a peak corresponding to 18 Da loss in molecular mass
occurred (Figure 2A, b), and when both ECH1 and ECH2 were
employed, two peaks corresponding to 18 and 62 Da loss in
molecular mass were observed (Figure 2A, d). These results suggest
that ECH1 catalyzes dehydration of HMG-ACP 5 to 3-methylgluta-
conyl-ACP 6 (Scheme 1), and ECH2 catalyzes subsequent decar-
boxylation to 7.
Next, we also demonstrated that ECH1 and ECH2 are able to
accept and catalyze dehydration and decarboxylation of (R,S)-HMG-
CoA (Figure 2B), which suggests that the two enzymes recognize
the phosphopantetheine arm from CoA as well as holo-ACP.
Importantly, this finding facilitated structural identification of the
dehydration and decarboxylation products from the reaction.
Specifically, 0.5 mM (R,S)-HMG-CoA was incubated with the
ECH1, ECH2, or both (2 µM each), in 30 mM bis-Tris buffer, pH
6.5 at 37 °C for 3 h, and the reaction mixtures were analyzed by
HPLC equipped with a photodiode array detector and an ESI-LTQ
mass spectrometer (ThermoFinnigan). UV spectral analysis of the
HMG-CoA substrate, and the following dehydration and decar-
boxylation steps, revealed a significant absorption increase at 260
nm for both dehydration and decarboxylation products (see Sup-
Supporting Information Available: Experimental details and
supplementary data. This material is available free of charge via the
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