Characterization of AntB, a promiscuous acyltransferase involved in antimycin biosynthesis

Article abstract of DOI:10.1021/ol4014365

The in vivo and in vitro characterization of AntB, a dedicated acyltransferase encoded in the antimycin biosynthetic gene cluster, which catalyzes the C-8 acyloxy formation is reported. It is demonstrated that AntB has broad substrate specificity toward both the acyl substrate and the acyl carrier and produces more antimycin analogues with varying C-8 acyloxy moieties.

Full text of DOI:10.1021/ol4014365

ORGANIC
LETTERS
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Vol. XX, No. XX
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Characterization of AntB, a Promiscuous
Acyltransferase Involved in Antimycin
Biosynthesis
Moriah Sandy, Xuejun Zhu, Zhe Rui, and Wenjun Zhang*
Department of Chemical and Biomolecular Engineering and Energy Biosciences
Institute, University of California, Berkeley, California 94720, United States
wjzhang@berkeley.edu
Received May 21, 2013
ABSTRACT
The in vivo and in vitro characterization of AntB, a dedicated acyltransferase encoded in the antimycin biosynthetic gene cluster, which catalyzes
the C-8 acyloxy formation is reported. It is demonstrated that AntB has broad substrate specificity toward both the acyl substrate and the acyl
carrier and produces more antimycin analogues with varying C-8 acyloxy moieties.
Antimycinsare a familyofnatural products containing a
nine-membered dilactone ring substituted with one alkyl
(C-7), one acyloxy (C-8), two methyl moieties (C-4 and
C-9), and an amide linkage (C-3) to a 30-formamidosa-
licylic acid (Figure 1). They are well-known for their
inhibitory action against the mitochondrial electron trans-
port chain resulting in significant biological activities includ-
ing antifungal, insecticidal, and nematocidal properties.1ꢀ3
Antimycin-type compounds have also been shown to sup-
press the production of pro-inflammatory protein cytokines
with minimal mammalian cell cytotoxicity, rendering them
as drug candidates for asthma treatment.4 In addition,
antimycins have great potential as anticancer drugs due
to their potent and selective inhibition of antiapoptotic
proteins Bcl2/BclXL.5ꢀ7 An antimycin derivative, 20-meth-
oxy antimycin A3, has been shown to bind Bcl2/BclXL and
trigger apoptosis without the general toxic effect on mito-
chondrial function.5,6 Computational modeling studies
predict that antimycins bind to the hydrophobic groove
of Bcl2/BclXL proteins, and the hydrophobicity of the side
chains, especially the C-8 acyloxyl group on the dilactone
ring, might directly affect the binding affinity and potency
of antimycins.5,6,8 Understanding the enzymatic mechan-
ism for antimycin biosynthesis will therefore promote the
production of more antimycin analogues with improved
pharmaceutical properties.
We recently dissected the antimycin biosynthetic path-
way by both genetic and enzymatic studies.9 A minimum
set of enzymes were identified to generate the antimycin
dilactone core using a nonribosomal peptide synthetase
(NRPS)-polyketide synthase (PKS) assembly line from
four distinct monomers: an aminobenzoate, a natural
amino acid, an R-keto acid, and an acylmalonyl moiety
(Figure 1). One presumable postassembly modification
step, a transesterification reaction to yield a C-8 acyloxy,
remains to be elucidated in order to generate the fully
(1) Dunshee, B. R.; Leben, C.; Keitt, G. W.; Strong, F. M. J. Am.
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Yamaguchi, Y.; Masuma, R.; Tomoda, H.; Chiba, T.; Yan, H.Kitamura,
Y.; Sugiura, W.; Omura, S.; Tanaka, H. J. Antibiot. 2005, 58, 65.
(4) Strangman, W. K.; Kwon, H. C.; Broide, D.; Jensen, P. R.;
Fenical, W. J. Med. Chem. 2009, 52, 2317.
(5) Schwartz, P. S.; Manion, M. K.; Emerson, C. B.; Fry, J. S.;
Schulz, C. M.; Sweet, I. R.; Hockenbery, D. M. Mol. Cancer Ther. 2007,
6, 2073.
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Zimmerberg, J.; Zhang, K. Y.; Hockenbery, D. M. Nat. Cell Biol. 2001,
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K. Y.; Hockenbery, D. M. J. Biol. Chem. 2004, 279, 2159.
(9) Sandy, M.; Rui, Z.; Gallagher, J.; Zhang, W. ACS Chem. Biol.
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10.1021/ol4014365
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modified antimycin dilactone ring. This tailoring step is
proposed to be catalyzed by AntB, a highly conserved
protein encoded in all identified antimycin biosyn-
thetic gene clusters.10,11 AntB shows moderate sequence
similarity to putative acyltransferases such as PldC12
(Identity/Similarity = 35%/50%) and LadG (Identity/
Similarity = 31%/46%) involved in the biosynthesis of
pladienolide and laidlomycin, respectively.
of antimycins that could be identified from the culture of
wild-type strain. Instead, a new set of metabolites were
detected in S. albus ΔantB supernatant extracts, with UV
absorbance spectra characteristic of antimycin-type com-
pounds, but more hydrophilic retention times compared
to those of the antimycins produced by the wild-type
strain (Figure 1). LC-High Resolution Mass Spectrometry
(HRMS) and HRMS/MS analysis established that these
new compounds are antimycin analogues with a C-8
hydroxyl moiety (Figures S8ꢀS11). Large-scale purifica-
tion of 6 was further performed for molecular struc-
ture characterization through NMR analysis (Tables S2,
S3ꢀS7). 6 was revealed to be a known antimycin-
type compound, uranchimycin B, which was previously
identified in marine actinomycetes.13 The accumulation of
compounds 4ꢀ7 suggested the role of AntB in the transes-
terification reaction to yield a C-8 acyloxyl substituent on
the dilactone core of antimycins. Although the reduction of
the C-8 β-keto to a hydroxy is catalyzed by AntM on
the AntD-tethered intermediate before its release from
the NRPS-PKS assembly line,9 the comparable yields
(∼10 mg/L) of 4ꢀ7 to those of the wild-type antimycins
strongly indicate that AntB catalyzes the transesterification
reaction after dilactone scaffold assembly.
The activity of AntB was then reconstituted in vitro to
further confirm the role of AntB as an acyltransferase.
antB from S. albus was amplified and cloned into the
expression vector pET-30 Xa/LIC, encoding an N-terminal
His6-tag. The corresponding protein was overproduced
in E. coli cells and purified using Ni-NTA affinity chro-
matography (Figure S1). Purified 6 was used as the acyl
acceptor, and the commercially available isobutyl-CoA
was chosen as the acyl donor because this same acyl
group occurs naturally at the C-8 position of the known
antimycins A2 (1) and A4 (3) (Figure 1).5,9,14 HPLC and
LC-HRMS/MS analysis of the enzymatic reactions
showed that AntB catalyzed the regioselective C-8 trans-
esterification of 6 to form 8 (Figures 2, S12). We next
examined the tolerance of AntB toward different acyl
moieties. A suite of antimycin-type compounds with pre-
sumably different C-8 side chains have been found to be
produced by S. albus, indicative of AntB possessing broad
substrate specificity toward the acyl substrate (Figure 1).9,10
Different acyl-CoAs as listed in Figure 2 were tested as
possible substrates for AntB in the biochemical assays.
AntB was shown to take all of the tested acyl-CoA sub-
strates except stearoyl-CoA. These results indicate that
AntB is fairly flexible toward the substituents on the acyl
moiety as long as the acyl groups are small enough to fit in
the AntB active site (Figures 2, S13ꢀS17).
Figure 1. (A) NRPS-PKS catalyzed antimycin biosynthesis. (B)
HPLC analysis (320 nm) of S. albus extracts showing production
of antimycins from the wild-type (lower trace) and not from
ΔantB (upper trace) and production of C8-hydroxy antimycins
from ΔantB but not from the wild-type.
In order to identify the dedicated enzyme responsible for
the transesterification on the antimycin C-8 hydroxyl
group, we first performed a gene disruption experiment
of antB in the antimycin-producing organism Streptomyces
albus J1074. antB was deleted in-frame through double
crossover, and the resulting mutant was confirmed by
PCR (Figure S2). High Performance Liquid Chroma-
tograpy (HPLC) analysis of culture extracts revealed that
the deletion of antB completely abolished the production
In addition to acyl groups, the substrate tolerance of
AntB toward an alternative acyl carrier N-acetylcystea-
mine (SNAC) was also tested. Using SNAC as an acyl
carrier increases the number of acyl substrates that can be
assayed, facilitated by the ease of chemical synthesis of
(10) Yan, Y.; Zhang, L.; Ito, T.; Qu, X.; Asakawa, Y.; Awakawa, T.;
Abe, I.; Liu, W. Org. Lett. 2012, 14, 4142.
(11) Seipke, R. F.; Barke, J.; Brearley, C.; Hill, L.; Yu, D. W.; Goss,
(13) Imamura, N.; Nishijima, M.; Adachi, K.; Sano, H. J. Antibiot.
1993, 46, 241.
R. J. M.; Hutchings, M. I. PLoS One 2011, 6, e22028.
(12) Machida, K.; Arisawa, A.; Takeda, S.; Tsuchida, T.; Aritoku,
Y.; Yoshida, M.; Ikeda, H. Biosci. Biotechnol. Biochem. 2008, 72, 2946.
(14) Kinoshita, M.; Aburaki, S.; Umezawa, S. J. Antibiot. 1972,
25, 373.
B
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acyl-SNACs compared to their CoA derivatives. Fur-
thermore, compared to acyl-CoAs, acyl-SNACs are cell-
permeable and therefore have potential use for precursor
feeding experiments in vivo.15,16 Several acyl-SNACs were
synthesized according to standard protocols and tested as
substrates for AntB.16 All of the acyl-SNACs were utilized
by AntB as shown in Figure 2 yielding the expected
products, which were confirmed by HPLC and HRMS/
MS analysis (Figures S12, S16, S18, S19). To date, no
antimycin-type compound containing an alkyne moiety
has been identified. We therefore carried out a large-scale
biocatalytic reaction with AntB, 6, and 4-pentynoyl-
SNAC. The resulting product, 15, was purified by HPLC,
and its molecular structure was confirmed by NMR
analysis (Figures S21ꢀS24, Table S3).
The kinetic parameters of AntB toward several acyl-CoAs
and acyl-SNACs were then determined using a 5,50-dithio-
bis(2-nitro-benzoic acid) (DTNB) spectrophotometric assay
to monitor the formation of free thiol in solution (Figure 3,
Table 1). AntB showed comparable Km values toward both
acyl carriers, while demonstrating significantly lower kcat
numbers for acyl-SNACs compared to acyl-CoAs, suggest-
ing that the shortening of the acyl carrier does not signifi-
cantly affect the substrate binding to the enzyme, albeit the
turnover rate is lowered by ∼100-fold. It is notable that
various acyl moieties tested in our assays have little effect on
the kinetic parameters of AntB, as the specificity constants
(kcat/Km) of AntB toward different acyl-SNACs were on the
same order of magnitude (Table 1). Even though kinetic
analysis of AntB demonstrated an obvious preference for
the CoA carrier, AntB can still effectively utilize acyl-SNAC
substrates in vitro with high overall percent conversions
(Table 1). In fact, both benzoyl-SNAC and isobutyl-SNAC
have higher percent conversions (95%, 42.3%) than their
CoA derivatives (81.1%, 17.3%) (Table 1), possibly due to
the instability of acyl-CoAs, or the hydrolysis of products in
the enzymatic reaction mixture. We further performed
precursor-directed biosynthesis by feeding 4-pentynoyl-
SNAC to wild-type cultures of S. albus. As expected, the
corresponding antimycin analogue was produced by S. albus
(Figure S25), demonstrating the feasibility of generating
new antimycin analogues using acyl-SNACs in vivo.
Figure 3. Determination of AntB kinetic parameters by the
DTNB assay. Error bars represent standard deviations from
at least three independently performed experiments. Each
assay was performed with a no-enzyme control, and the rate
of spontaneous thioester hydrolysis was subtracted in each
case.
Figure 2. HPLC analysis (320 nm) showing in vitro production
of antimycin analogues with variations at the C8 position
catalyzed by AntB. R1 groups in 8, 12, and 14 are found in
naturally produced antimycins.10 Reaction conditions: 50 mM
HEPES, pH 8.0, 1 mM 6, 1 mM acyl-CoA or acyl-SNAC, 10 μM
AntB, 25 °C, 2 h. *The corresponding acyl-CoA or acyl-SNAC
was also tested and was shown to yield the same product.
In summary, we have established AntB as an acyltransfer-
ase responsible for tailoring the antimycin dilactone scaffold
at the C-8 hydroxy by transesterification. We have demon-
strated that AntB has relaxed substrate specificity, and
in vitro assays with AntB and a variety of acyl substrates
resulted in the production of several new antimycin
(15) Jacobsen, J. R.; Hutchinson, C. R.; Cane, D. E.; Khosla, C.
Science 1997, 277, 367.
(16) Xie, X.; Watanabe, K.; Wojcicki, W. A.; Wang, C. C.; Tang, Y.
Chem. Biol. 2006, 13, 1161.
Org. Lett., Vol. XX, No. XX, XXXX
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further derivatizations.17 Our characterization of AntB,
along with our previous characterization of the other en-
zymes required for antimycin dilactone scaffold assembly,
provides a superior platform for priming the antimycin
enzymatic machinery to generate new antimycin analogues
for drug development.
Table 1. Acylthioester Substrates of AntBa
Acknowledgment. We thank B. Shen (TSRI) for pro-
viding S. albus J1074 and J. Pelton (UC Berkeley) for
helping with NMR spectroscopic analysis. This work was
supported by the Pew Scholars Program and University
of California Cancer Research Coordinating Committee
funds.
Supporting Information Available. Experimental Meth-
ods, SDS-PAGE analysis of purified AntB, NMR char-
acterization of 6 and 15, LCMS and LCMS/MS analysis
of compounds 4ꢀ15. This material is available free of
charge via the Internet at http://pubs.acs.org.
a Reactions were performed in 50 mM HEPES pH 8 using 10 μM
AntB, 1 mM 6, 4 mM acyl-thioester, 10 h. The products of the reactions
were verified by HPLC, HRMS, and HRMS/MS. b HPLC (320 nm) was
used to calculate the percent of 6 converted to the corresponding
antimycin analogues. The data were the averages from three indepen-
dently performed experiments.
(17) Dupuis, S. N.; Robertson, A. W.; Veinot, T.; Monro, S. M. A.;
Douglas, S. E.; Syvitski, R. T.; Goralski, K. B.; McFarland, S. A.;
Jakeman, D. L. Chem. Sci. 2012, 3, 1640.
analogues with varying C-8 acyloxyl groups. The incorpora-
tion of a 4-pentynoyl moiety in 15 is particularly interesting,
presenting a unique method to install a chemical handle for
The authors declare no competing financial interest.
D
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