Identification of middle chain fatty Acyl-CoA Ligase responsible for the biosynthesis of 2-alkylmalonyl-CoAs for Polyketide extender unit

Article abstract of DOI:10.1074/jbc.M115.677195

Background: Fatty acyl-CoA ligases involved in polyketide biosynthesis remain uncharacterized. Results: RevS classified in fatty acyl-AMP ligase clade was the middle chain fatty acyl-CoA ligase. Conclusion: RevS was responsible for 2-alkylmalonyl-CoA bios

Full text of DOI:10.1074/jbc.M115.677195

JBC Papers in Press. Published on September 16, 2015 as Manuscript M115.677195
The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M115.677195
Middle chain fatty acyl-CoA ligase involved in reveromycin A biosynthesis
Identification of middle chain fatty acyl-CoA ligase responsible for the biosynthesis of
2
-alkylmalonyl-CoAs for polyketide extender unit
a,b
a,1
b
a
a
Takeshi Miyazawa , Shunji Takahashi , Akihiro Kawata , Suresh Panthee , Teruo Hayashi ,
a
a
a,b,1
Takeshi Shimizu , Toshihiko Nogawa , and Hiroyuki Osada
a
RIKEN Center for Sustainable Resource Science, Chemical Biology Research Group, Saitama 351-0198,
Japan
b
Graduate School of Science and Engineering, Saitama University, Saitama 338-8570, Japan
Running title: Middle chain fatty acyl-CoA ligase involved in reveromycin A biosynthesis
1To whom correspondence should be addressed. E-mail: shunjitaka@riken.jp, hisyo@riken.jp
Key words: Biosynthesis, enzyme catalysis, secondary metabolite, natural product, polyketide
Other key words: Acyl-CoA ligase, alkylmalonyl-CoA, reveromycin, Streptomyces
Background:
reintroduction of the gene restored the yield of
RMs. Although RevS was classified in the fatty
acyl-AMP ligase (FAAL) clade based on
Fatty acyl-CoA ligases involved in polyketide
biosynthesis remain uncharacterized.
Result:
phylogenetic
analysis,
biochemical
RevS classified in fatty acyl-AMP ligase clade was
middle chain fatty acyl-CoA ligase
Conclusion:
RevS was responsible for 2-alkylmalonyl-CoA
biosynthesis through enzyme coupling with RevT
reductase/carboxylase.
characterization revealed that the enzyme
catalyzed middle chain fatty acyl-CoA ligase
(FACL) but not FAAL activity, suggesting the
molecular evolution for acyl-CoA biosynthesis.
Moreover, we examined the in vitro conversion of
fatty acid into 2-alkylmalonyl-CoA using purified
RevS and RevT. The coupling reaction showed
efficient conversion of hexenoic acid into
butylmalonyl-CoA. RevS efficiently catalyzed C8-
C10 middle chain FACL activity, therefore, we
speculated that the acyl-CoA precursor was
truncated via β-oxidation and converted into (E)-2-
enoyl-CoA, a RevT substrate. To determine
whether the β-oxidation process is involved
between RevS and RevT reaction, we performed
Significance:
2
-Alkylmalonyl-CoA biosynthesis was strongly
supported by the function of RevR and RevS,
which utilized fatty acids derived from de novo
biosynthesis
respectively.
and
degradation
products,
Abstract
Understanding the biosynthetic mechanism of the
atypical polyketide extender unit is important to
develop bioactive natural products. Reveromycin
1
3
the feeding experiment using [1,2,3,4- C]octanoic
1
3
acid. C NMR analysis clearly demonstrated
1
3
(
RM)-derivatives produced by Streptomyces sp.
incorporation of the [3,4- C]octanoic acid moiety
into the structure of RM-A. Our results provide
insight into the role of uncharacterized RevS
homologs that may catalyze middle chain FACL to
produce a unique polyketide extender unit.
SN-593 possess several aliphatic extender units.
Here, we studied the molecular basis of 2-
alkylmalonyl-CoAformation by analyzing the revR
and revS genes, which form a transcriptional unit
with the revT gene,
a
crotonyl-CoA
Introduction
reductase/carboxylase homolog. We mainly
focused on uncharacterized adenylate-forming
enzyme (RevS). revS gene disruption resulted in
the reduction of all RM-derivatives, while
Microorganisms harbor structurally diverse
natural products, including polyketides, peptides,
and terpenoids (1). Their secondary metabolites
with strong biological activity have been utilized as
1
Copyright 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Middle chain fatty acyl-CoA ligase involved in reveromycin A biosynthesis
antibiotics, antitumor drugs, immunosuppressants,
apoptosis in osteoclasts (34). The activity was also
linked to the inhibition of osteolytic bone
metastasis induced by lung and prostate cancer
cells (34-37). Streptomyces sp. SN-593 produces
various RM-derivatives, including RM-A (1), -C
(2), -D (3), and -E (4) (Fig. 1A), whose structural
diversity is derived from incorporation of atypical
extender units such as butylmalonyl-CoA,
isobutylmalonyl-CoA, pentylmalonyl-CoA, and
hexylmalonyl-CoA, respectively. In our previous
study, we identified 1 biosynthetic gene cluster
possessing RevT, a CCR homolog (38). Although
the presence of the revT gene strongly suggested
the production of 2-alkylmalonyl-CoA for RM
biosynthesis, we identified the revR and revS genes,
which are associated in the same transcriptional
unit (Fig. 1B) (38). Moreover, each gene homolog
was also found in several biosynthetic gene clusters
that produce polyketide compounds, including the
atypical extender unit (Fig. 1) (27-32). Thus, we
predicted an important role for the production of 2-
alkylmalonyl-CoA by RevR and RevS, whose
genes were annotated to be -ketoacyl-[acyl carrier
protein (ACP)] synthase III (KASIII) and
adenylate-forming enzyme family, respectively.
In this study, we examined the molecular basis
of 2-alkylmalonyl-CoA formation for RM
biosynthesis through gene disruption, kinetic
analysis, and precursor feeding experiments. Our
results indicated that RevR is important for
selective production of 1 via de novo fatty acid
synthesis, and that RevS may represent a novel
class of FACLs associated with the fatty acid
degradation process, providing insight into the
dynamics of fatty acid metabolism for polyketide
biosynthesis. Moreover, the identification of RevS
function sheds light on unexplained RevS
homologs found in other polyketide biosynthetic
gene clusters.
hypercholesterolemia drugs, and insecticides (2).
Unique chemical structures are linked to a variety
of biological activities, therefore, understanding
the biosynthetic machinery is important for future
combinatorial biosynthesis (3-5).
Fatty acyl chains found in microbial natural
products are responsible for biological activity and
physiological function in microorganism. For
instance, daptomycin binds to the cell membranes
of Gram-positive bacteria through its lipid moiety,
followed by calcium-dependent insertion and
oligomerization (6). Dimerization of alkyl
resorcinol results in the generation of potent
proteasome inhibitors, cylindrocyclophanes (7).
The fatty acyl chain of mycolic acid plays an
important role in membrane construction in
Mycobacterium tuberculosis (8).
Fatty acyl-AMP ligase (FAAL), a member of the
adenylate-forming enzyme superfamily (9),
catalyzes ATP-dependent formation of acyl-AMP
and loading onto the phosphopantetheine arm of
acyl carrier protein (ACP). FAALs are associated
with the polyketide synthase (PKS) and
nonribosomal peptide synthetase (NRPS) gene
clusters, and the resulting fatty acyl-ACP is
incorporated into the starter unit of the PKS and
NRPS assembly line (7,10-21). In contrast to
FAAL, fatty acyl-CoA ligase (FACL) catalyzes
ATP-dependent conversion of fatty acid into fatty
acyl-CoA through an acyl-AMP intermediate. The
activated fatty acyl-CoA is utilized for
phospholipid synthesis, energy generation,
transcriptional regulation, and protein acylation
(
22,23). However, involvement of FACL in the
supply of available fatty acyl-CoA for PKS
extender unit remains unclear. Atypical extender
units are incorporated into polyketide structures
through a variety of biosynthetic pathways (24-32).
Among the related enzymes, crotonyl-CoA
carboxylase/reductase (CCR) homologs which
catalyze reductive carboxylation of ,-
unsaturated acyl-CoA/ACP have been extensively
characterized as an essential step (Fig. 1A).
Experimental procedures
Chemicals
and
Enzymes―Ampicillin,
kanamycin, chloramphenicol, and CoA were
purchased from Nacalai Tesque, Inc. (Kyoto,
Japan). Streptomycin, spectinomycin, thiostrepton,
ATP, NADH, NADPH, Tris (2-carboxyethyl)
phosphine (TCEP), myokinase, pyruvate kinase,
Reveromycin A (RM-A) is a polyketide
compound produced by Streptomyces sp. SN-593
(
33). It inhibited bone resorption by inducing
2

Middle chain fatty acyl-CoA ligase involved in reveromycin A biosynthesis
and lactate dehydrogenase were purchased from
were utilized.
Sigma-Aldrich (St. Louis, MO, USA). All fatty
acids used in this study were purchased from Tokyo
Chemical Industry Co., Ltd. (Tokyo, Japan).
Carumonam was purchased from Takeda
Pharmaceutical Co., Ltd. (Tokyo, Japan). [1-
Bacterial Strains and Plasmids―Escherichia
coli DH5 (Takara Bio Inc., Shiga, Japan) and E.
coli GM2929 hsdS::Tn10 carrying pUB307-
aph::Tn7 were used for general DNA manipulation
and E. coli-Streptomyces sp. SN-593 conjugation,
1
3
13
C]Hexanoic acid and [1,2,3,4- C]octanoic acid
TM
were purchased from Cambridge Isotope
Laboratories, Inc. (Cambridge, MA, USA). All
commercially available chemicals for chemical
synthesis were used without further purification.
respectively (39). Escherichia coli BL21 Star
(DE3) (Invitrogen, Carlsbad, CA, USA) and
Streptomyces lividans TK23 were to prepare the
recombinant protein, and pET28b(+) (Novagen,
Madison, WI, USA) and pWHM3 (40) were used
as expression vectors, respectively.
Analytical Methods―The H and 13C NMR
1
spectra of 1 and CoA derivatives were recorded on
JEOL
JNM-ECA-500
and
JNM-AL-400
Culture Conditions―E. coli strains were grown
at 37C in LB (1% Bacto-tryptone (BD
Biosciences, Franklin Lakes, NJ, USA), 0.5% yeast
extract (BD Biosciences), and 1% NaCl), LB agar
Miller (Nacalai Tesque, Inc., Kyoto, Japan), or
Terrific broth (TB) (1.2 % (w/v) Bacto-tryptone,
spectrometers (JEOL, Ltd., Tokyo, Japan).
Chemical shifts (in ppm) were referenced against
the residual solvent for H NMR and TSP-d4 as an
1
external standard for 13C NMR. UV absorbance
was measured using a JASCO V-630 BIO
spectrophotometer (JASCO Co., Tokyo, Japan).
Fast atom bombardment (FAB) mass spectra were
obtained using a JMS-700 mass spectrometer.
Electrospray ionization (ESI) mass spectra of CoA-
derivatives were obtained on a Bioapex-II Fourier
transform ion cyclotron resonance mass
spectrometer (Bruker Daltonics Japan, Ltd., Tokyo,
Japan) and Synapt G2 (Waters, MA, USA). ESI-
MS analysis of RM derivatives and RevS and RevT
reaction products were performed using a Waters
Alliance HPLC system equipped with a mass
spectrometer (Q-TRAP, Applied Biosystems, CA,
USA)
2
0
K
1

.4% (w/v) yeast extract, 0.4% (v/v) glycerol,
.231% (w/v) KH PO , and 1.254% (w/v)
HPO ). To select for plasmid-containing cells,
00 g ml ampicillin, 50 g ml kanamycin, 30
2
4
2
4
-1
-1
-1
-1
g ml chloramphenicol, 50 g ml streptomycin,
-1
or 100 g ml spectinomycin were added to an LB
plate. Streptomyces sp. SN-593 culture conditions
were described previously (38). S. lividans TK23
cells were cultured at 28C in SK2 medium (1.4%
(
(
w/v) soluble starch, 0.35% (w/v) glucose, 0.35%
w/v) yeast extract, 0.21% (w/v) Bacto-peptone,
0
0
.21% (w/v) beef extract (Remel Inc., KS, US),
.014% (w/v) KH PO , and 0.042% (w/v) MgSO
2
4
4
;
Matrix-assisted laser desorption/ionization-
time-of-flight (MALDI-TOF)/mass spectrometry
pH 7.0).
(
MS) analysis of acyl-ACP was performed using
Plasmid Construction for Gene Disruption and
Complementation―The revR, revS, and revT gene
disruption was performed by PCR-targeted gene
replacement using the plasmids pKD46, pKD13,
and pCP20 and E. coli BW25113 (41,42).
Ampicillin-resistant pKD46 containing the λ Red-
mediated recombination functions was used with
the chloramphenicol-resistant pCC1FOS fosmid
clone 3C11 containing the revR, revS, and revT
genes. The plasmid pKD13 was used as a template
for the FRT-flanked kanamycin-resistant gene
cassette (42,43).
ultrafleXtreme (Bruker Daltonics, Billerica, MA,
USA). Sinapinic acid was used as a matrix.
All chemical syntheses were carried out under a
nitrogen atmosphere and monitored by TLC with
0
5
was achieved using UV light and 10% ethanol
solution of phosphomolybdic acid, followed by
heating. For column chromatography, Silica Gel 60
N (spherical, neutral, 0.04–0.05 mm. Kanto
Chemical Co, Inc., Tokyo, Japan) and Cosmosil
.25-mm pre-coated silica gel plates 60F254 Art
715 (Merck, Darmstadt, Germany). Visualization
7
5C18-DNP (Nacalai Tesque Inc., Kyoto, Japan)
3

Middle chain fatty acyl-CoA ligase involved in reveromycin A biosynthesis
To construct the revR, revS, and revT gene
for 10 sec and 25 cycles of 98C for 10 sec, 62C
for 5 sec, and 68C for 30 sec (45). The primers
used for amplification are shown in Table 1. After
restriction enzyme digestion, the revS, revT,
SRE2849, and sfp gene fragments were inserted
into the NdeI and XhoI sites of pET28b(+) or
pACYCDuet-1 to construct pET28b-revS,
replacement plasmids, DNA fragments containing
the disrupted gene were amplified by PCR using

PrimeSTAR HS DNA polymerase (TaKaRa Bio).
PCR conditions were as follows: 98C for 10 sec,
5 cycles of 98C for 10 sec, 62C for 5 sec, and
8C for 5 min. The amplified fragments were
2
6
pET28b-revT,
pET28b-SRE2849,
and
ligated to the HindIII site of the pIM vector (38).
For Southern analysis (Fig. 2), the AlkPhos Direct
Labelling and Detection System (GE Healthcare,
Little Chalfont, UK) was used. The primers used
for gene disruptions and Southern analysis are
shown in Table 1.
To obtain the gene complementation plasmid,
the revR, revS, and revT genes were ligated into the
BamHI and HindIII sites of pTYM19 with aphII
promoter (38,44). Because the revR gene includes
a BamHI site in the sequence, a silent mutation was
introduced using the QuikChange protocol
pACYCDuet-1-sfp, respectively.
Plasmid Construction for Heterologous Gene
Expression in S. lividans TK23―The β lactamase
gene in the pWHM3 vector was replaced with the
aphI gene using λRed recombination to form pWK.
Next, the tipA promoter (PtipA) was inserted into the
EcoRI and BamHI sites to construct pWK-PtipA for
expression in S. lividans TK23. To facilitate
enzyme purification, the revT gene containing a
His8-tag coding sequence was amplified from
pET28b-revT using the primers shown in Table 1.
After restriction enzyme digestion, the fragment
was inserted into the BamHI and HindIII sites of
pWK-PtipA to construct pWK-PtipA-revT.
(
Stratagene, La Jolla, CA, USA). The revR gene
inserted into the NdeI/XhoI restriction sites of the
pET28b vector was amplified using PfuTurbo DNA
polymerase (Stratagene) and the following primer
pairs (with the mutation sites underlined): 5′-
TCCAGCGACGAGTGGATACGCCGGCACTC
Heterologous Gene Expression and Purification
of Enzymes―To obtain recombinant RevS, the
CGGGAT-3′
and
5′-
expression plasmid was transformed into E. coli
ATCCCGGAGTGCCGGCGTATCCACTCGTCG
CTGGA-3′. PCR conditions were as follows: 98C
for 30 sec, 20 cycles of 98C for 30 sec, 60C for 1
min, and 68C for 13 min. The resultant pET28b-
revR-BamHI_mut vector was used as a template for
the complementation plasmid (Table 1).
TM
BL21 Star
(DE3) cells. The resultant
transformants were grown on LB medium
-1
containing 50 g ml kanamycin overnight. To 200
-1
ml TB containing 50 g ml kanamycin, the
preculture was added at an initial optical density of
0.1 at 600 nm (OD600). Cells were cultured at 18C.
When the OD600 reached 0.6, isopropyl β-D-1-
thiogalactopyranoside was added to a final
concentration of 0.5 mM. After further growth for
18 h at 18C, the cells were harvested by
centrifugation and frozen at –80C.
Plasmid Construction for Heterologous Gene
Expression in E. coli―The revS (1740 bp) and revT
(
1332 bp) genes were amplified from the fosmid
®
clone (PCC1FOS-3C11) using PrimeSTAR HS
DNA polymerase under the following conditions:
9
6
8C for 10 sec and 25 cycles of 98C for 10 sec,
2C for 5 sec, and 68C for 1.5 min. The SRE2849
gene (243 bp) coding ACP of Streptomyces sp. SN-
93 was amplified from PCC1FOS-4B1 under the
following conditions: 98C for 10 sec and 25 cycles
of 98C for 10 sec, 60C for 5 sec, and 68C for 25
sec. The Bacillus subtilis 168 phosphopantetheinyl
transferase gene (sfp) (654 bp) was amplified from
pUC19-sfp under the following conditions: 98C
After thawing on ice, the cells were suspended
in 20 ml of extraction buffer (50 mM Tris-HCl, pH
7
.5, 500 mM NaCl, and 20% glycerol, 5 mM
-
1
-
imidazole, 0.5 mg ml lysozyme, and 6.25 U ml
5
1
benzonase (Sigma-Aldrich)). For stabilization of
recombinant RevS, octanoic acid was added to the
cell suspension at a final concentration of 1 mM.
The cell suspension was sonicated 10 times on ice
for 10 sec with 1 min interval between each
4

Middle chain fatty acyl-CoA ligase involved in reveromycin A biosynthesis
sonication treatment (UD-200, TOMY, Tokyo,
Japan). Cell debris was removed by centrifugation
10,000 ×g for 30 min) (SRX-201, TOMY), and
then the supernatant was applied to a Ni-
nitrilotriacetic acid (Ni-NTA) agarose (Qiagen,
Hilden, Germany) column (2 × 4 cm) that had been
equilibrated with buffer A (50 mM Tris-HCl, pH
transformed into E. coli BL21 StarTM (DE3) cells.
The transformants were selected on LB medium
-1
(
containing 50 g ml kanamycin. Heterologous
expression and purification were performed as
described for RevS. The fraction eluted from the
Ni-NTA column was concentrated using an
Amicon Ultra centrifugal filter, and the buffer was
exchanged for stock buffer.
7
5
.5, 500 mM NaCl, and 20% glycerol) containing
mM imidazole. After washing with the same
To obtain holo-ACP, pET28b-SRE2849 and
pACYCDuet-1-sfp were co-transformed into E.
buffer, non-specifically bound proteins were
removed by washing with 20 ml of buffer A
containing 5 mM imidazole and 0.2% Tween 20.
After removing Tween 20 with 40 ml of buffer A
containing 5 mM imidazole, the column was
further washed with 40 ml of buffer A containing
TM
coli BL21 Star (DE3) cells. Transformants were
-
1
selected on LB medium containing 50 g ml
-1
kanamycin and 30 g ml chloramphenicol.
Heterologous gene expression, Ni-NTA column
chromatography, and buffer exchange were
performed as described for apo-ACP. To eliminate
40 mM imidazole. The His-tag fusion protein was
eluted with 25 ml of butter A containing 250 mM
imidazole. The eluted fraction was concentrated
using an Amicon Ultra centrifugal filter (Merck,
Darmstadt, Germany), and the buffer was
exchanged for stock buffer (50 mM Tris-HCl, pH

-N-gluconoylated holo-ACP (48) from the Ni-
NTA fraction, holo-ACP was further purified using
a MonoQ 5/50 column (GE Healthcare) that had
been pre-equilibrated with 50 mM Bis-Tris (pH
6.5) buffer containing 0.2 M NaCl and 20%
7.5, 200 mM NaCl, 20% glycerol) on the
glycerol. After loading the Ni-NTA fraction (42.2
mg), the column was washed with equilibration
buffer for 30 min at a flow rate of 0.2 ml min , and
centrifugal filter. The purity of RevS was
confirmed by SDS-PAGE. Size exclusion
chromatography was performed as described
previously (46).
To obtain recombinant RevT, pWK-PtipA-revT
was introduced into S. lividans TK23 using a
polyethylene glycol-mediated protoplast method
-1
eluted with a linear gradient of 0.2–0.5 M NaCl for
60 min. Subsequently, 400 l of the Mono Q
fraction (5.7 mg) containing holo-ACP was
collected based on MALDI-TOF/MS analysis and
used in the FAAL assay for RevS.
(
47). The resulting transformants were grown in
-1
SK2 medium containing 15 μg ml kanamycin for
FACL Assay of RevS－For HPLC analysis of the
RevS reaction product, an enzyme reaction was
performed in a final reaction volume of 100 l
containing 100 mM HEPES (pH 7.5), 20% glycerol,
2
days, and then 1 ml preculture was inoculated
-1
into 70 ml of SK2 medium containing 15 μg ml
kanamycin. After 1 day of culture, thiostrepton was
-1
added at a final concentration of 50 μg ml to
express RevT. After another 1 day of culture, the
cells were harvested by centrifugation and frozen
at –80C. Purification of RevT was performed as
described for RevS. The His-tag fusion protein was
eluted with 5 ml of butter A containing 250 mM
imidazole and 1 mM NADPH and used for
biochemical characterization. The purity of RevT
was confirmed by SDS-PAGE. Size exclusion
chromatography was performed using 50 mM Tris-
1
0 mM MgCl , 5 mM TCEP, 0.5 mM fatty acid, 2
2
mM CoA, 3 mM ATP, and 1 M RevS. After
preincubation of the reaction mixture at 25C for 3
min, the reaction was initiated by the addition of
RevS and allowed to proceed for 60 min. The
reaction was terminated by the rapid addition of 0.5

l formic acid and then centrifuged at 15,000 ×g
for 10 min at 4C (Kubota 3700, Kubota Co.,
Tokyo, Japan) to remove the protein. The
supernatant
chromatography (LC)/ESI- MS (Table 2).
To determine the substrate specificity of RevS,
we quantified fatty acyl-CoA formation by HPLC
was
subjected
to
liquid
HCl (pH 7.5), 10 mM NaHCO
0% glycerol.
To obtain apo-ACP, pET28b-SRE2849 was
3
, 200 mM NaCl, and
1
5

Middle chain fatty acyl-CoA ligase involved in reveromycin A biosynthesis
and NADH oxidation using a spectrophotometer.
4 h. The reaction mixture (10 l) was applied to
Microcon Ultracel YM-10 (Millipore, MA, USA).
After buffer exchange with 50 mM Tris-HCl (pH
7.5), the formation of (E)-2-nonenoyl-ACP was
confirmed based on MALDI-TOF/MS analysis.
The reaction mixture (70 l) containing (E)-2-
nonenoyl-ACP was used for RevT reaction.
Because the stoichiometry of the product amount
in both assays was identical, we performed a
spectrophotometric assay to determine the kinetic
parameters of RevS (49). Assays were performed
in a 1-ml quartz cuvette with a final volume of 200

l containing 100 mM HEPES (pH 7.5), 20%
glycerol, 10 mM MgCl , 5 mM TCEP, 0.05–3 mM
2
fatty acid, 2 mM CoA, 3 mM ATP, 0.4 mM
phosphoenol pyruvate, 0.4 mM NADH, 5 U
myokinase, 4.5 U pyruvate kinase, 5.8 U lactate
dehydrogenase, and 0.5–2.5 M RevS. Substrate
and RevS concentrations were varied as follows:
Enzyme Assay of RevT―To detect the RevT
reaction product, the assay were performed in a
final volume of 100 l containing 100 mM Tris-
3
HCl (pH 7.5), 10% glycerol, 10 mM NaHCO , 11
mM MgCl , 1 mM EDTA, 0.5 mM (E)-2-enoyl-
2
0
.1–2 mM heptanoic acid with 2 M RevS, 0.1–2
CoA, 4 mM NADPH, and 1 M RevT. After
preincubation of the reaction mixture at 25C for 3
min, the reaction was initiated by the addition of
RevT and allowed to proceed for 10–30 min. The
reaction was terminated by the rapid addition of 0.5
mM octanoic acid with 1 M RevS, 0.2–2 mM
nonanoic acid with 1 M RevS, 0.1 to 2 mM
decanoic acid with 2 M RevS, 0.2–2.2 mM (E)-2-
hexenoic acid with 2 M RevS, 0.1–2.5 mM (E)-2-
heptenoic acid with 2.5 M RevS, 0.1–3 mM (E)-

l formic acid and centrifuged at 15,000 ×g for 10
min at 4C to remove the protein. The supernatant
was subjected to LC/ESI-MS analysis.
2-octenoic acid with 2 M RevS, 0.2–2.5 mM (E)-
2-nonenoic acid with 1 M RevS, 0.05–3 mM (E)-
2-decenoic acid with 2 M RevS, 0.05–3 mM (E)-
2-undecenoic acid with 2 M RevS, 0.05–3 mM
To determine the kinetic parameters of RevT, we
quantified both butylmalonyl-CoA formation by
HPLC and NADPH oxidation using
a
(
(
(
(
E)-3-hexenoic acid with 1 M RevS, 0.05–3 mM
E)-3-heptenoic acid with 1 M RevS, 0.05–3 mM
E)-3-octenoic acid with 0.5 M RevS, 0.05–3 mM
E)-3-nonenoic acid with 0.5 M RevS, and 0.05–
spectrophotometer. Because the stoichiometry of
the reaction product in both assays was identical,
we conducted a spectrophotometric assay. The
assay was performed in a 1-ml quartz cuvette in a
final volume of 200 l containing 100 mM Tris-
3
mM (E)-3-decenoic acid with 0.5 M RevS. The
cofactor concentration varied from 0.01–0.1 mM
for ATP and from 0.02–1 mM for CoA. After
preincubation of the reaction mixture at 25C for 3
min, the reaction was initiated by adding RevS and
the initial rate of oxidation of NADH at 340 nm
was measured using a spectrophotometer. The
kinetic constant was calculated by a nonlinear
regression fit to the Michaelis–Menten equation
using SigmaPlot11 (Table 3).
HCl (pH 7.5), 10% glycerol, 10 mM NaHCO
3
, 11
mM MgCl , 1 mM EDTA, 0.5 mM (E)-2-enoyl-
2
CoA, 0.3 mM NADPH, and 1 M RevT. NADPH
concentration was varied from 0.01–0.3 mM. The
substrate concentration for (E)-2-enoyl-CoAvaried
from 0.1–2 mM. After preincubation of the
reaction mixture at 25C for 3 min, the reaction was
initiated by adding RevT, and NADPH oxidation at
340 nm was measured using a spectrophotometer.
The kinetic constant was calculated by a nonlinear
regression fit to the Michaelis–Menten equation
using SigmaPlot11 (Table 4).
Preparation of (E)-2-nonenoyl-ACP―(E)-2-
nonenoyl-ACP was prepared from a reaction
containing 100 mM HEPES (pH 7.5), 20% glycerol,
To examine the substrate specificity of RevT for
1
0 mM MgCl , 5 mM TCEP, 0.5 mM (E)-2-
2
(
E)-2-enoyl-ACP, the reaction was performed in a
final volume of 40 l containing 100 mM Tris-HCl
pH 7.5), 10% glycerol, 10 mM NaHCO , 11 mM
MgCl , 1 mM EDTA, 0.23 mM (E)-2-nonenoyl-
ACP, 4 mM NADPH, and 5 M RevT. After the
nonenoic acid, 0.4 mM of holo-ACP, 3 mM ATP,
and 5 M RevS in a final volume of 80 l. After
preincubation at 25C for 3 min, the reaction was
initiated by the addition of RevS and incubation for
(
3
2
6

Middle chain fatty acyl-CoA ligase involved in reveromycin A biosynthesis
reaction was allowed to proceed at 25C for 3 h, the
been equilibrated with 2% solvent B, the column
was developed over a linear gradient of 2–25%
solvent B for 30 min and washed with 100%
solvent B for 10 min. Mass spectra were collected
in the ESI-negative mode.
reaction mixture (40 l) was applied to Microcon
Ultracel YM-10. After buffer exchange with 50
mM Tris-HCl (pH 7.5), the reaction product was
analyzed by MALDI-TOF/MS.
Feeding of Labeled Precursors and Isolation of
1―The revR mutants were grown in 70 ml SY
medium for 2 days at 28C, and then 1 ml of the
preculture was inoculated into 70 ml of PV8
medium. After 2 days of culture at 28C, [1-
In Vitro Reconstruction of Butylmalonyl-
CoA―An enzyme coupling assay was performed
for 1 h at 25C in 100 mM Tris-HCl (pH 7.5) buffer
containing 20% glycerol, 10 mM NaHCO
3
, 11 mM
MgCl , 1 mM EDTA, 5 mM TCEP, 0.5 mM (E)-2-
2
1
3
13
hexenoic acid, 2 mM CoA, 3 mM ATP, 4 mM
NADPH, 1 M RevS, and 1 M RevT in a final
volume of 100l. The reaction was terminated by
the rapid addition of 0.5 l formic acid and
centrifuged at 15,000 ×g for 10 min at 4C to
remove the protein. The supernatant was subjected
to LC/ESI-MS analysis.
C]hexanoic acid or [1,2,3,4- C]octanoic acid
were added to the culture at a final concentration of
.3 mM. Five days after inoculation, an equal
0
volume of acetone was added to the broth and
filtered to remove the mycelia. The filtrate was
evaporated in vacuo to obtain an aqueous solution.
The solution was adjusted to pH 4 using acetic acid
and extracted twice with the same volume of ethyl
acetate. The organic layer was dried over Na
and concentrated under reduced pressure to yield a
brown oil. 1.5 g of extract from 2 L of [1-
2
SO
4
LC/ESI-MS Analysis of Enzyme Reaction
Products―To analyze of the reaction products of
RevS, a LC/ESI-MS analysis was carried out. An
Applied Biosystems Q-TRAP was connected to a
Waters Alliance 2965 with a 2996 photodiode array
detector and an XTerraMS C18 column (5 m, 2.1
1
3
C]hexanoic acid feeding culture and 0.5 g of
1
3
extract from 1 L of [1,2,3,4- C]octanoic acid
feeding culture were obtained.
The 1.5 g extract from [1- C]hexanoic acid
feeding was subjected to silica gel chromatography
with stepwise-elution from 100:0 to 0:100 with
1
3
×
150 mm (Waters)). The HPLC conditions were as
-1
follows: 0.25 ml min flow rate; solvent A: water
containing 40 mM ammonium acetate (pH 6.8);
solvent B: acetonitrile. The conditions of the linear
gradient elution were optimized to detect each
RevS reaction product. The column was
equilibrated with 2% solvent B. After injection of
CHCl
3
–methanol. 1 was eluted 100:10 with CHCl -
3
methanol. The fraction was separated using a
Waters 600 HPLC system equipped with a 2996
photodiode array detector and a Senshu Pak Pegasil
ODS column (20 × 250 mm) using an
acetonitrile/0.05% aqueous formic acid isocratic
1
μl of the sample into the equilibrated column, the
column was developed using a linear gradient from
–20% for saturated acyl-CoA, 2–50% for (E)-2-
-1
system (48/52) at a flow rate of 9 ml min to yield
.7 mg of 1.
The 0.5 g extract from the [1,2,3,4- C]octanoic
2
7
enoyl-CoA, and 2–65% acetonitrile for (E)-3-
enoyl-CoA over 45 min. After the gradient, the
column was washed with 100% solvent B for 10
min. All mass spectra were collected in the ESI-
negative mode (Table 2).
1
3
acid feeding was separated on a Sephadex LH-20
column (GE Healthcare) and eluted with methanol.
The fractions containing 1 were applied to a 12-g
RediSep Rf column (Teledyne Isco, Inc., Lincoln,
NE, USA) for medium-pressure liquid
chromatography with linear gradient from 20:80 to
To analyze the reaction products of RevT,
LC/ESI-MS analysis was carried out. The HPLC
conditions were as follows: 5 m (2.1 × 150 mm)
1
00:0 of ethyl acetate-hexane for 35 min at a flow

-1
column, XTerra MS C18; 0.25 ml min flow rate;
solvent A: water containing 40 mM ammonium
acetate (pH 5); solvent B: acetonitrile. After
injection of 1 μl sample into the column that had
-1
rate 30 ml min to obtain 28 fractions. The fraction
containing 1 was separated on a Senshu Pak Pegasil
ODS (10
×
250 mm) column using
a
7

Middle chain fatty acyl-CoA ligase involved in reveromycin A biosynthesis
methanol/0.05% aqueous formic acid isocratic
system (67/33) at a flow rate of 4 ml min to yield
1020.0952;
found:
1020.0952,
[M+Na] , 998.1133; found:
998.1132. H NMR (500 MHz, D O) δ: 0.76 (s, 3H),
.88 (t, J = 6.9 Hz, 3H), 0.91 (s, 3H), 1.30 (m, 4H),
-1
+
+
C H N
28 43 7
O
19
1
P
3
SNa
4
1.6 mg of 1.
2
0
Preparation
of
Butylmalonyl-
1.84 (m, 2H), 2.46 (t, J = 6.9 Hz, 2H), 3.07 (m, 2H),
3.38 (m, 2H), 3.48 (m, 2H), 3.57 (m, 2H), 3.87 (dd,
J = 9.8, 4.6 Hz, 1H), 4.04 (s, 1H), 4.27 (brs, 2H),
4.62 (brs, 1H), 6.21 (d, J = 6.9 Hz, 1H), 8.31 (s,
CoA―Butylmalonyl-CoA was prepared using the
transesterification method (50). To a stirred
solution of butylmalonic acid (2.11 g, 13.2 mmol)
and thiophenol (1.42 g, 12.8 mmol) in 100 ml of
dry N,N-dimethylformamide (DMF), was added a
solution of N,N'-dicyclohexylcarbodiimide (DCC)
3.20 g, 15.5 mmol) in 100 ml of DMF dropwise at
C. After stirring for 2 h at 0C, the reaction was
quenched with 50 ml of cold water and the mixture
was acidified to pH 4 using 1 M HCl. The
precipitated solid was filtered off and the filtrate
was extracted 3 times with 200 ml of ether. The
combined ether layers were dried over Na
concentrated. The residue was purified by silica gel
column chromatography (n-hexane/ethyl acetate;
/1) to give monothiophenyl butylmalonate as a
colorless solid (465 mg, 1.86 mmol, 14%). The Rf
value was 0.1 (n-hexane/ethyl acetate; 8/3). H
1
3
1H), 8.59 (s, 1H). C NMR (125 MHz, D O) :
2
16.00, 20.76, 23.75, 24.64, 30.88, 31.90, 31.92,
32.22, 32.31, 38.20, 38.28, 41.12, 41.19, 41.48,
66.98, 68.39, 68.42, 74.73, 74.78, 76.84, 76.84,
76.93, 76.93, 86.67, 86.73, 89.26, 121.52, 142.78,
152.31, 155.83, 158.56, 176.78, 177.59, 179.02,
203.85.
(
0
Preparation of (E)-2-hexenoyl-CoA and (E)-2-
octenoyl-CoA―(E)-2-hexenoyl-CoA was also
prepared using the transesterification method (50).
To a stirred solution of (E)-2-hexenoic acid (1.14 g,
10 mmol), thiophenol (10 g, 9.0 mmol), and N, N-
dimethyl-4-aminopyridine (20 mg) in 10 ml of dry
methylene chloride, was added a solution of DCC
(2.48 g, 12.0 mmol) in 5 ml of dry methylene
chloride at 0C. After stirring for 1 h at 0C and
then for 1 h at room temperature, the precipitated
solid was filtered off and the organic layer was
evaporated. The residue was purified using silica
gel column chromatography (n-hexane/ethyl
acetate, 10/1) to give thiophenyl (E)-2-hexenoate
as an oil (1.54 g, 7.46 mmol, 74%). The Rf value
2
SO and
4
1
1
NMR (400 MHz, CDCl
3
) 0.93 (t, J = 7.0 Hz, 3H)
.39 (m, 4H), 2.01 (m, 2H), 3.70 (t, J = 7.6 Hz, 1H),
.43 (m, 5H). HRMS (FAB) m/z calculated for
1
7
C
-
H O
13 15 3
S [M-H] , 251.0742; found: 251.0739. To
a stirred solution of CoA (free form, 100 mg, 130.2
mmol) in 7.6 ml of 0.1 M NaHCO , was added
3
monothiophenyl butylmalonate (164 mg, 651
mmol, 5eq) at 0°C, and then 0.2 M NaOH solution
was slowly added to increase the pH to 8. After
stirring overnight, the solution was acidified to pH
1
was 0.3 (n-hexane/ethyl acetate; 5/1). H NMR
(
(
400 MHz, CDCl
tq, J = 7.2, 7.2 Hz, 1H), 2.22 (dtd, J = 7.2, 7.2, 1.2
3
) : 0.98 (t, J = 7.2 Hz, 3H) 1.53
5
using approximately 2 ml of 0.2 M HCl. The
mixture was fractionated with 5 ml of ether twice,
and 5 ml of ethyl acetate to remove excess
thiophenyl ester and thiophenol. The resulting
aqueous phase was lyophilized and the residue
Hz, 2H), 6.19 (dt, J = 15.6, 1.2 Hz, 1H), 6.99 (dt, J
15.6, 7.2 Hz, 1H), 7.44 (m, 5H). To a stirred
solution of CoA (free form, 50 mg, 65.1 mmol) in
.8 ml of 0.1 M NaHCO solution, was added the
solution of thiophenyl (E)-2-hexenoate (67.2 mg,
25.5 mmol, 5 eq) in 3 ml of tetrahydrofuran at 0C.
After stirring overnight at 4C, the reaction mixture
was concentrated and fractionated with 5 ml of
ether twice to remove excess thiophenyl ester and
thiophenol. The resulting aqueous phase was
lyophilized and the pH of the residue was adjusted
=
3
3
(
220 mg), after adjusting the pH to 7.4 with 0.1 M
NaHCO solution, was purified using Cosmosil
3
3
column chromatography (gradient elution with
water/methanol; methanol: 0–7.5%). A combined
fraction was lyophilized to give butylmalonyl-CoA
in amorphous powder (101 mg, 111 mmol, 85%
yield based on free form). The Rf value was 0.3 (n-
butanol/acetic acid/H
calculated for C28
2
O; 4/1/2). HRMS (ESI) m/z
to 7.4 using 0.1 M NaHCO
3
solution before charge.
+
+
H
42
N
7
O
19
P
3
SNa
5
[M+Na] ,
The residue was purified using Cosmosil column
8

Middle chain fatty acyl-CoA ligase involved in reveromycin A biosynthesis
chromatography
(gradient
solution
with
adjusting the pH to 7.4 with 0.1 M NaHCO
3
water/methanol; methanol: 0–12.5%). A combined
fraction was lyophilized to give (E)-2-hexenoyl-
CoA in amorphous powder (40 mg, 46.3 mmol,
solution before charge, was purified using
Cosmosil column chromatography (gradient
elution with water/methanol; methanol: 0–20%). A
combined fraction was lyophilized to give (E)-2-
octenoyl-CoA in amorphous powder (24 mg, 26.9
mmol, 41% yield based on free form). The Rf value
7
0
1% yield based on free form). The Rf value was
.3 (n-butanol/acetic acid/H O; 4/1/2). HRMS
2
+
(
[
C
9
0
2
2
2
ESI) m/z calculated for C27
M+Na] ,
H
found:
41
N
7
O
17
P
3
SNa
952.1081,
[M+Na] , 930.1264; found:
30.1264. H NMR (500 MHz, D O) δ: 0.77 (s, 3H),
4
+
952.1083;
was 0.3 (n-Butanol/ acetic acid/H
HRMS (ESI) m/z calculated
[M+Na] , 980.1396; found:
2
O; 4/1/2).
+
+
H
27 42
N
7
O
17
1
P
3
SNa
3
for
+
+
2
C
29
H
45
N
7
O
17
P
3
SNa
4
+
+
.91 (t, J = 6.3 Hz, 3H), 0.91 (s, 3H), 1.48 (m, 2H),
.20 (dq, J = 6.9, 1.7 Hz, 2H), 2.46 (t, J = 6.3 Hz,
H), 3.07 (t, J = 6.3 Hz, 2H), 3.39 (t, J = 6.9 Hz,
H), 3.47 (t, J = 6.9 Hz, 2H), 3.58 (dd, J = 9.8, 4.6
980.1393,
958.1577; found: 958.1570. H NMR (500 MHz,
O) δ: 0.77 (s, 3H), 0.87 (t, J = 6.9 Hz, 3H), 0.91
29
C H
46
N
7
O
17
P
3
1
SNa
3
[M+Na] ,
D
2
(s, 3H), 1.29 (m, 4H), 1.45 (m, 2H), 2.21 (m, 2H),
2.46 (t, J = 6.9 Hz, 2H), 3.08 (t, J = 6.3 Hz, 2H),
3.39 (t, J = 6.3 Hz, 2H), 3.47 (t, J = 6.3 Hz, 2H),
3.58 (dd, J = 9.8, 5.2 Hz, 1H), 3.86 (dd, J = 9.8, 4.6
Hz, 1H), 4.05 (s, 1H), 4.26 (m, 2H), 4.60 (brs, 1H),
6.21 (m, 2H), 6.98 (dt, J = 15.4, 6.9 Hz, 1H), 8.29
Hz, 1H), 3.86 (dd, J = 9.7, 4.6 Hz, 1H), 4.05 (s, 1H),
.27 (m, 1H), 4.60 (brs, 1H), 6.21 (m, 2H), 6.98 (dt,
4
1
3
J = 15.4, 6.9 Hz, 1H), 8.29 (s, 1H), 8.59 (s, 1H). C
NMR (125 MHz, D O) : 15.78, 20.97, 23.43,
3.73, 30.66, 36.59, 38.59, 38.26, 38.30, 41.15,
2
2
4
7
1
1
1
3
1.21, 41.54, 68.64, 68.68, 74.69, 74.73, 76.52,
6.55, 76.96, 77.35, 86.97, 87.01, 89.43, 121.51,
30.75, 142.80, 151.50, 152.32, 155.80, 158.55,
76.86, 177.59, 196.77.
(s, 1H), 8.59 (s, 1H). C NMR (125 MHz, D O) :
2
16.12, 20.98, 23.74, 24.60, 29.62, 30.66, 33.44,
34.48, 38.27, 38.31, 41.15, 41.22, 41.53, 68.63,
68.67, 74.69, 74.73, 76.51, 76.54, 76.96, 77.36,
77.38, 86.95, 86.99, 89.43, 121.50, 130.60, 142.79,
(
E)-2-octenoyl-CoA was synthesized from (E)-
151.87, 152.30, 155.79, 158.53, 176.85, 177.58,
196.76.
2
-octenoic acid using the mixed anhydride method
(
(
51-53). To a stirred solution of (E)-2-octenoic acid
92.6 mg, 651 mmol) and triethylamine (81.4 mg,
Results
8
13 mmol, 1.25 eq) in 20 ml of dry ether, was
added ethyl chloroformate (88.2 mg, 813 mmol,
.25 eq) slowly at 0C. After stirring for 12 h at
Disruption of RevR, RevS, and RevT Genes
and Analysis of Metabolite Profiles－In the 1 gene
cluster, the revR, revS, and revT genes were in the
same transcriptional unit. Based on BLAST
searching, revT gene belongs to the CCR homologs
which are distributed in gene clusters producing
polyketide compounds with atypical extender units
(27-31). In addition to the revT gene, homologs of
the revR and revS genes were also distributed in
various polyketide gene clusters (Fig.1) (I, II, III,
and IV). Therefore, to understand the role of revR,
revS, and revT genes, we conducted gene
disruptions (Figs. 2A, 2C, and 2E). After
confirmation of gene disruption (revR, revS,
revT) by Southern hybridization (Fig. 2B, 2D, and
2F), the metabolite profile of each gene disruptant
was analyzed by LC/ESI-MS (Fig. 3). In the wild-
type strain, 1 was the major product among the
RM-derivatives (Fig. 1A and 3A). Interestingly,
1
room temperature under an argon atmosphere, the
separated solid was filtered off and washed quickly
with a small amount of dry ether. The whole
solution containing an approximately 10-fold
molar excess of CoA as a reactant (ether solution,
2
0 ml: 32.55 M) was stored at 0C under argon.
To a stirred solution of CoA (free form, 50 mg,
6
5.1 mmol) in 4 ml of 0.05 M Na
2
CO solution, 3
3
ml of ethyl acetate and 3 ml of ethanol, an ether
solution of 2 ml of 65.1 mmol crude ethyl 1-oxo-
oct-2-en carbonate (n-hexane/ethyl acetate; 5/3; Rf
=
0.3) was added dropwise at 0C at pH 8.2. After
stirring for 2 h at room temperature, the reaction
mixture was evaporated under reduced pressure to
remove the organic solvent, and the resulting
aqueous phase was lyophilized. The residue, after
9

Middle chain fatty acyl-CoA ligase involved in reveromycin A biosynthesis

revR mutants selectively decreased the
purified the protein to homogeneity using Ni-NTA
column chromatography (Fig. 4D). Because the
His-tag was not attached in Sfp, only holo-ACP
production of 1 (Fig. 3B). Reintroduction of the
revR gene under the control of the aph promoter
restored 1 production to wild-type levels (Fig. 3C).
was
purified
using Ni-NTA column

revS mutants showed an approximately 50%
chromatography after co-expression of Sfp and
ACP in E. coli. The holo-ACP was further
purified by MonoQ column chromatography and
its modification was confirmed by MALDI-
TOF/MS analysis (Fig. 4E). Next, purified RevS
was incubated with various fatty acids in the
presence of ATP and holo-ACP. Although the
FAAL and FACL activities of RevS showed the
same substrate specificity, the specific activity of
FAAL was significantly lower than that of FACL
reduction of all RM-derivatives (Fig. 1A and 3D),
and the phenotype was recovered by reintroduction
of the revS gene (Fig. 3E), suggesting that RevS is
involved in the formation of C18 alkyl residues in
RM derivatives. Additionally, revT mutants
completely abolished the production of RMs (Fig.
3F), which was restored by complementation with
the revT gene (Fig. 3G), indicating that RevT is
essential for the production of RMs.
(
Fig. 4F).
Purification
and
Characterization
of
RevS―Based on BLAST searching, RevS belongs
to the uncharacterized adenylate-forming enzyme
family. Together with the gene disruption
phenotype (Fig. 3D and 3E), we hypothesized that
RevS should function as an FACL or FAAL. We
heterologously expressed His8-tagged RevS in E.
coli and purified the protein to homogeneity using
Ni-NTA column chromatography (Fig. 4A). Both
monomeric (65 kDa) and dimeric (123 kDa) RevS
were observed following gel filtration analysis (Fig.
Analysis of Kinetic Properties of FACL activity
of RevS―To determine the kinetic parameters of
RevS for each substrate, we performed
spectrophotometric assays. The RevS reaction
apparently followed Michaelis-Menten kinetics.
High catalytic efficiency was observed when
middle chain fatty acids (C8-C10) were used for
the RevS assay (Table 3). In addition, RevS
accepted both saturated and unsaturated fatty acids
as substrates with the same catalytic efficiency. We
also calculated kinetic constants for ATP and CoA.
4B). After gel filtration chromatography, we
evaluated the specific activity of the monomeric
and dimeric fractions. Based on the FACL assay,
the specific activities of the monomer and dimer
fractions of RevS were the same as that of the
purified RevS before gel filtration (Fig. 4C).
Therefore, the multimeric status of RevS on gel
filtration chromatography was speculated to be the
monomer-dimer equilibrium.
The K
m
and kcat values for ATP were 0.039 ± 0.004
-1
mM and 56.7 ± 2.2 min , respectively, giving a
catalytic efficiency of 1456 min mM . The K
and kcat values of CoA were 0.05 ± 0.01 mM and
-1
-1
m
-1
39.6 ± 0.8 min , respectively, giving a catalytic
-1
-1
efficiency of 861 min mM . The kinetic
parameters were comparable to those for other
FACLs reported previously (Table 3).
To examine FACL activity, purified RevS was
incubated with various fatty acids in the presence
of ATP and CoA and the reaction products were
analyzed by LC/ESI-MS. RevS activated both
saturated and unsaturated fatty acids with carbon
chain lengths of C4–C11 into fatty acyl-CoAs
Phylogenetic Analysis of RevS―To obtain
insight into the molecular evolution of RevS, we
performed phylogenetic analysis of the RevS using
the amino acid sequences of FACL, FAAL, and a
variety of CoA ligases (Fig. 5A). Consistent with
the presence of an FAAL-specific insertion motif
(Fig. 5B) (14), RevS was classified in an FAAL
clade. However, biochemical analyses clearly
demonstrated that RevS is an FACL. Interestingly,
RevS homologs such as CinT and SamR0482
associated in the biosynthetic gene cluster of
(Table 2), but did not activate fatty acids with chain
lengths of more than C13. Efficient acyl-CoA
formation was observed when middle chain fatty
acids (C8-C10) were used as the substrate.
Moreover, to examine FAAL activity, we
heterologously expressed holo-ACP in E. coli and
1
0

Middle chain fatty acyl-CoA ligase involved in reveromycin A biosynthesis
cinnabaramide and stambomycin (Fig. 1B),
efficiently converted fatty acids into octanoyl-CoA,
nonanoyl-CoA, and decanoyl-CoA, which contain
longer carbon chain than physiological RevT
substrates. Therefore, the -oxidation of fatty acyl-
CoAs is essential for producing truncated (E)-2-
enoyl-CoAs for RevT substrates. To understand the
biosynthetic process, we performed a feeding
respectively, were found in the same branch,
suggesting that CinT and SamR0482 may be
middle chain FACLs. These RevS homologs may
have evolved from the FAAL family and were
adapted for the production of fatty acyl-CoAs to
efficiently convert them into 2-alkylmalonyl-CoAs
for the PKS extender unit.
1
3
experiment using [1,2,3,4- C]octanoic acid. We
hypothesized the activation of labeled fatty acid
1
3
Purification and Biochemical Characterization
of RevT―To determine the function of RevT, His8-
tag RevT was heterologously expressed in S.
lividans TK23 and purified using Ni-NTA column
chromatography. The molecular mass of the RevT
was estimated to be 48 kDa by SDS-PAGE (Fig.
into [1,2,3,4- C]octanoyl-CoA by RevS, the
1
3
generation of [1,2- C](E)-2-hexenoyl-CoA after

-oxidation process, and the conversion into [1,2-
C]butylmalonyl-CoA by RevT, resulting in
1
3
assembly into the structure of 1 (Fig. 7A). In this
experiment, we used the revR mutant for efficient
incorporation of the fatty acid precursor into 1,
6
(
A) and 178 kDa by gel filtration chromatography
Fig. 6B), suggesting that RevT exists as a tetramer.
Incubation of purified RevT in the presence of (E)-
-hexenoyl-CoA, NaHCO , and NADPH was
because the mutants showed low yields of 1 (Fig.
13
3
B). After feeding of [1,2,3,4- C]octanoic acid,
2
3
13
we isolated 1 and analyzed its C-NMR spectrum.
In addition to the signals at 25.4 and 84.2 ppm
assigned to C17 and C18 of 1, respectively,
significant C-C coupling was observed. Moreover,
the coupling constants were nearly the same value
performed for 30 min. The time-dependent
production of butylmalonyl-CoA was observed for
20 min (Fig. 6C).
Because the understanding of substrate
specificity and kinetic properties of RevT is
important for the synthesis of polyketide
compounds, we also characterized the kinetic
parameters of (E)-2-hexenoyl-CoA and (E)-2-
octenoyl-CoA. The reaction apparently followed
Michaelis-Menten kinetics. RevT catalyzed both
(
J = 36 and 37 Hz) (Fig. 7B). These results suggest
that octanoic acid was incorporated into 1 through
the biosynthetic pathway predicted in Fig. 7A. We
also tested whether [1- C]hexanoic acid is
incorporated into 1 (Fig. 7C). After the feeding
experiment, we purified 1 from the revR mutant
culture and its C-NMR spectrum was analyzed
(
assigned to C17 of 1 was highly enriched,
suggesting that hexanoic acid is utilized for 1
biosynthesis.
1
3
(
E)-2-hexenoyl-CoA and (E)-2-octenoyl-CoA with
13
the same catalytic efficiency. The K and kcat values
m
Fig. 7D). A corresponding signal at 25.4 ppm
of (E)-2-hexenoyl-CoA for the formation of
butylmalonyl-CoA were 0.33 ± 0.06 mM and 29.9
-
1
±
1.7 min , respectively, giving a catalytic
-1
-1
efficiency of 91.4 min mM . The K
m
and kcat
values of (E)-2-octenoyl-CoA for the formation of
RevS and RevT Are Responsible for
Butylmalonyl-CoA Biosynthesis―Because of
efficient incorporation of fatty acids into 1, we also
investigated the successive conversion of (E)-2-
hexenoic acid into (E)-2-hexenoyl-CoA by RevS
and the reductase/carboxylase reaction by RevT to
give butylmalonyl-CoA (Fig. 8A). As expected,
hexylmalonyl-CoA were 0.33 ± 0.04 mM and 36.2
-1
±
1.3 min , respectively, giving a catalytic
-1
-1
efficiency of 109 min mM . The kinetic
parameters were also evaluated for NADPH in the
presence of (E)-2-hexenoyl-CoA and (E)-2-
octenoyl-CoA (Table 4). The kinetic efficiency of
RevT for (E)-2-octenoyl-CoA was better than that
of CinF (31). Catalytic efficiency of SalG was
significantly higher than that of RevT (27).
(
E)-2-hexenoic acid was efficiently converted into
butylmalonyl-CoA in the RevS-RevT coupling
reaction (Fig. 8B). This is the first report of in vitro
reconstruction of 2-alkylmalonyl-CoA using
physiological enzymes derived from a secondary
13
Incorporation of C Labeled Fatty Acids into
1
―Biochemical analysis indicated that RevS
11

Middle chain fatty acyl-CoA ligase involved in reveromycin A biosynthesis
metabolite gene cluster.
the involvement of unidentified CoA ligase, which
Moreover, we examined whether the
biosynthetic route occurs from (E)-2-hexenoic acid
to (E)-2-enoyl-ACP and then 2-alkylmalonyl-ACP
may support medium chain acyl-CoA formation.
We found three genes showing 30–43% amino acid
identity to RevS in the draft genome of
Streptomyces sp. SN-593. Although three were not
in the FACL clade of RevS (Fig. 5A), gene
disruption and biochemical analysis will be
essential for final conclusions.
(
Fig. 8C). Because the FAAL activity of RevS was
very low (Fig. 4F), we first purified the FAAL
reaction product, (E)-2-nonenoyl-ACP, using
MonoQ column chromatography. Using the
purified (E)-2-nonenoyl-ACP fraction, we
performed the RevT reaction under optimized
condition (Fig. 6). However, we did not detect the
conversion of (E)-2-nonenoyl-ACP into 2-
heptylmalonyl-ACP (Fig. 8D), suggesting that
RevS and RevT are responsible for the production
of 2-alkylmalonyl-CoA but not 2-alkylmalonyl-
ACP.
Overall, structure of the class I adenylate-
forming enzymes are composed of large N-
terminal and small C-terminal domains connected
by a flexible hinge region (9). Crystal structure
analysis of FAAL28 (FadD28) from M.
tuberculosis revealed that FAAL has a specific
insertion motif containing 22 amino acids (Fig. 5B).
The insertion motif modulates C-terminal domain
movement and disrupts access of the acyl-AMP
intermediate to the CoA binding motif (14). Amino
acid alignments and phylogenetic analysis of
adenylate-forming enzymes indicated that RevS
belongs to the FAAL clade. Unexpectedly,
biochemical analysis of RevS revealed FACL
activity even in the presence of the FAAL-specific
insertion motif (Fig. 5). Arora et al. also reported
that deletion of the insertion motif found in FadD28
resulted in the conversion of FAAL activity into
FACL activity (14), indicating that FAAL
originally contains CoA in the binding pocket.
Since phylogenetic analysis suggested that FAAL
was derived from CoA ligase, the FACL activity of
RevS may have re-evolved from the FAAL clade
by generating access to the CoA binding pocket by
optimizing the orientation of the N- and C-terminal
domains. Interestingly, in contrast to RevS,
FadD10, which does not possess the FAAL-
specific insertion motif, from M. tuberculosis
showed FAAL activity. This was explained by the
orientation of its N-terminal and C-terminal
domains, which are quite different from those of
other adenylate-forming family members (58).
Therefore, crystal structure analysis of RevS is
essential for fully understanding its biosynthetic
mechanism.
Discussion
In this study, we characterized the revR, revS,
and revT genes, which are involved in 2-
alkylmalonyl-CoA formation in RM biosynthesis.
revR gene disruption demonstrated that RevR was
involved in the selective production of
butylmalonyl-CoA, which was consistent with the
high production of 1 in wild-type culture (Fig. 3).
RevR showed 55% amino acid identity to BenQ
that is reported to be crucial for providing the
hexanoate PKS starter unit for benastatin
biosynthesis (54,55). In addition, it has been
reported that KASIII is a determinant of the fatty
acid priming unit producing straight or branched
chain, as well as the even- or odd number chain
(
56,57). RevR is likely to select butyryl-CoA as a
priming substrate to yield 3-oxo-hexanoyl-ACP.
Next, the product may be converted into (E)-2-
hexenoyl-ACP by -keto processing enzymes (Fig.
1C). In this de novo fatty acid biosynthetic pathway,
an unknown transacylase may be responsible for
the conversion of (E)-2-hexenoyl-ACP into (E)-2-
hexenoyl-CoA, as RevT did not accept (E)-2-
enoyl-ACP as a substrate (Fig. 8D). revS gene
disruption and complementation analysis indicated
the non-essentiality of RevS for the
biosynthesis of RMs (Fig. 3D and 3E). We
speculate that RMs biosynthesis in revS
mutants was mainly supported by de novo fatty
acid biosynthesis (Fig. 1C). Another possibility is
Metabolite profiling of revS gene disruptants
(
Fig. 2 and 3) and kinetic analysis of RevS (Fig. 4,
Table 2 and 3) also suggested the presence of
available free fatty acids (C6–C10) during the late
stationary phase and the close relationship between
1
2

Middle chain fatty acyl-CoA ligase involved in reveromycin A biosynthesis
fatty acid metabolism and polyketide biosynthesis.
acyl-ACP-thioesterases, Cp FatB1 and CP FatB2,
respectively, which is consistent with the
accumulation of middle chain fatty acids in the
seed (62). However, we found no homologous gene
in Streptomyces sp. SN-593. Additional detailed
studies are needed to understand fatty acid
Streptomyces typically utilizes de novo-
synthesized fatty acids not only for building
membrane phospholipids but also to accumulate
neutral lipid storage compounds such as
triacylglycerol (TAG) (59-61). TAG is one of
important carbon sources for energy production
and a precursor for secondary metabolism. A link
between TAG degradation and actinorhodin
production has been suggested (59). Because TAG
found in S. lividans consists of fatty acids ranging
from C14–C18 (59), the fatty acids derived from
TAG degradation should mainly be utilized for the
production of acetyl-CoA, but not as RevS
substrates. Therefore, other mechanisms may
generate medium chain fatty acids to support the
homeostasis
biosynthesis.
during secondary
metabolite
In summary, 2-alkylmalonyl-CoA biosynthesis
was strongly supported by the functions of RevR
and RevS. These enzymes effectively utilized de
novo fatty acid biosynthesis and fatty acid
degradation products, respectively. Decreased
supply of atypical building blocks in the polyketide
assembly line is critical for secondary metabolite
biosynthesis. Therefore, the presence of RevR and
RevS may be a backup system to ensure the
production of atypical extender units. Our results
explain why homologs of RevR and RevS are
distributed in polyketide biosynthetic gene clusters,
which utilize atypical extender units (Fig. 1).
RevS reaction.
termination of fatty acid biosynthesis by chain
length-specific acyl-ACP-thioesterase.
A
possible mechanism is
Interestingly, it has been reported that the seed of
Cuphea palustris possessed C8- and C14-specific
Acknowledgments—We thank Dr. K. Matsumoto for valuable advice and comments. We thank Dr. M.
Uramoto for NMR analysis. We thank Dr. N. Doumae for MALDI-TOF/MS analysis. We thank Dr. T.
Nakamura and Dr. Y. Hongo for HRMS analysis. We thank Dr. T. Kumano, E. Oowada, H. Takagi, and A.
Okano for technical assistance.
Conflict of interest: The authors declare that they have no conflicts of interest with the contents of this
article.
Author contributions: Takeshi Miyazawa performed the genetic, biochemical, and feeding experiments,
and wrote the paper. Shunji Takahashi designed all of the studies, constructed vectors, and wrote the paper.
Akihiro Kawata examined the assay condition for RevS. Suresh Panthee supported the isolation of gene
disruptants. Teruo Hayashi and Takeshi Shimizu synthesized butylmalonyl-CoA, (E)-2-hexenoyl-CoA, and
(
E)-2-octenoyl-CoA. Toshihiko Nogawa supported LC/MS and NMR analysis. Hiroyuki Osada integrated
this research. All authors reviewed the results and approved the final version of the manuscript.
1
3

Middle chain fatty acyl-CoA ligase involved in reveromycin A biosynthesis
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Acta 1521, 59-65
Khare, G., Gupta, V., Gupta, R. K., Gupta, R., Bhat, R., and Tyagi, A. K. (2009) Dissecting the
role of critical residues and substrate preference of a Fatty Acyl-CoA Synthetase (FadD13) of
Mycobacterium tuberculosis. PLoS One 4, e8387
Chang, C., Huang, R., Yan, Y., Ma, H., Dai, Z., Zhang, B., Deng, Z., Liu, W., and Qu, X. (2015)
Uncovering the Formation and Selection of Benzylmalonyl-CoA from the Biosynthesis of
Splenocin and Enterocin Reveals a Versatile Way to Introduce Amino Acids into Polyketide
Carbon Scaffolds. J Am Chem Soc 137, 4183-4190
1
9

Middle chain fatty acyl-CoA ligase involved in reveromycin A biosynthesis
FOOTNOTES
*
This work was partially supported by the JSPS KAKENHI Grant 24380052 and by a grant for "Project
focused on developing key technology of discovering and manufacturing drug for next-generation
treatment and diagnosis" from the Ministry of Economy, Trade and Industry (METI), Japan.
1
To whom correspondence may be addressed: RIKEN Center for Sustainable Resource Science, Chemical
Biology Group, Saitama 351-0198, Japan, Tel.: +81-48-467-4044; Fax: +81-48-462-4669; E-mail:
shunjitaka@riken.jp, hisyo@riken.jp
2
The abbreviations used are: ACP, acyl carrier protein; CCR, crotonyl-CoA carboxylase/reductase;
LC/ESI-MS, liquid chromatography/electrospray ionization mass spectrometry; FAAL, fatty acyl-
AMP ligase; FACL, fatty acyl-CoA ligase; NRPS, nonribosomal peptide synthetase; PKS, polyketide
synthase; RM, reveromycin
2
0

Middle chain fatty acyl-CoA ligase involved in reveromycin A biosynthesis
FIGURE 1. Biosynthetic genes responsible for polyketide extender units of RM-A. A, Polyketide natural
products containing atypical extender units. Bold in red indicates carbon chains derived from atypical
extender units. B, Gene organization. Based on the presence of the revR, revS, and revT homologs, each
gene cluster was categorized into four groups, including: revR, revS, and revT homologs (I), revR and revT
homologs (II), revT homolog with additional genes related to atypical extender unit biosynthesis (III), and
revS homolog with putative propionyl-CoA carboxylase -subunit homolog (IV). C, The scheme of the
possible 2-alkylmalonyl-CoA biosynthesis by RevR, RevS, and RevT. Both fatty acid degradation and de
novo fatty acid biosynthetic pathway were predicted to be involved in 2-alkylmalonyl-CoA biosynthesis.
FIGURE 2. Gene disruption and Southern hybridization. A, scheme of revR gene disruption and
restriction enzyme map of wild-type and ΔrevR gene mutant strains. The bar shows the expected fragment
size (bp) following NcoI and SalI digestion of genomic DNA. The bold bar shows the 3608-bp probe. B,
Southern hybridization of genomic DNA from wild-type and revR gene disruptants. C, scheme of revS gene
disruption and a restriction enzyme map of wild-type and ΔrevS gene mutant strains. The bar shows the
expected fragment size (bp) after ApaLI and BalI digestion of genomic DNA. The bold bar shows the 5180-
bp probe. D, Southern hybridization of genomic DNA from wild-type and revS gene disruptant. E, scheme
of revT gene disruption and restriction enzyme map of wild-type and ΔrevT gene mutant strains. The bar
shows the expected fragment size (bp) following EcoRI and NotI digestion of genomic DNA. The bold bar
shows 5399-bp probe. F, Southern hybridization of genomic DNA from wild-type and revT gene disruptant.
Closed and open triangles show DNA fragments from wild-type and mutant strains, respectively.
FIGURE 3. Metabolite profiles of gene disruptants. HPLC analysis of ethyl acetate extract from wild-
type strain (A), revR mutant (B), and revR mutant complemented with revR gene (C). revS mutant (D)
and revS mutant complemented with revS gene (E). revT mutant (F) and revT mutant complemented
with revT gene (G). 1–4 indicate RM-A, RM-C, RM-D, and RM-E, respectively. Asterisk indicates the peak
of RM-B, which was non-enzymatically converted into 5,6-spiroacetal structure from 1 (38).
FIGURE 4. Evaluation of FACL and FAAL activity of RevS. A, SDS-PAGE analysis of RevS using
12.5% polyacrylamide gel. Lane 1, molecular mass markers; lane 2, purified RevS (1 μg). B, size exclusion
chromatography of RevS. Purified protein (0.8 mg) was analyzed on a Superdex 200 column. C, Analysis
of specific activity of the RevS fractions after gel filtration. The purified RevS after Ni-NTA column
chromatography was fractionated by Superdex 200 chromatography (46). Two main peaks corresponding
to the monomer and dimer fractions (each 4 ml) were concentrated using an Amicon Ultra centrifugal filter.
In the presence of 1 M RevS, 1 mM of octanoic acid, and 2 mM CoA, the specific activity was calculated
using the spectrophotometric assay described in the Experimental procedures. Lane 1, purified RevS before
gel filtration; lane 2, monomer fraction of RevS after gel filtration; lane 3, dimer fraction of RevS after gel
filtration. D, SDS-PAGE analysis of purified apo- and holo-ACP using 15% polyacrylamide gel. Lanes 1
and 4, molecular mass markers; lane 2, His8-tag purified apo-ACP (1 μg); lane 3, His8-tag purified holo-
ACP (1 μg). E, MALDI-TOF/MS analysis of apo-ACP (i), and holo-ACP and -N-gluconoylated holo-
ACP fraction after Ni-NTA column chromatography (ii), purified holo-ACP after removing -N-
gluconoylated holo-ACP by MonoQ column chromatography (iii). F, FACL (open bar) and FAAL (closed
bar) activity of RevS were measured using an enzyme-coupled spectrophotometric assay. 0.3 mM of CoA
or 0.3 mM of the purified holo-ACP was used to calculate specific activity of RevS.
FIGURE 5. Phylogenetic analysis and multiple sequence alignments of RevS.
A, Phylogenetic analysis of functionally annotated FACL, FAAL, and CoA ligases. Multiple sequence
aliments were performed using the bootstrap method of CLASTALW (63). The bootstrap tree was drawn
2
1

Middle chain fatty acyl-CoA ligase involved in reveromycin A biosynthesis
using the neighbor-joining method in MEGA, version 6.06 (www.megasoftware.net). B, sequence aliments
of RevS with FACL and FAAL, whose crystal structures have been solved. The bold bar shows FAAL-
specific insertion motif (14,64).
Accession numbers used are as follows: Mav_Mig (AAB87139) from Mycobacterium avium; Ath
At4g05160 (Q9M0X9), Ath ACOS5 (AAP03019.1), Ath 4-coumarate-CoA ligase 1 (AAD47191), Ath 4-
coumarate-CoA ligase 2 (AAD47192), Ath 4-coumarate-CoA ligase 3 (AAD47194), and Ath 4-coumarate-
CoA ligase 4 (Q9LU36) from Arabidopsis thaliana; Sce Faa1p (P30624) and Sce Faa3p (P39002) from
Saccharomyces cerevisiae S288c, Rno FACL ACS1 (NP_036952), Rno ACS4 (NP_446075), Rno ACS5
(
NP_446059), Rno ACS6_v1 (P33124), Rno FATP1 (Q60714) and Rno FATP4 (Q91VE0) from Rattus
norvegicus; Eco FAAL (3PBK) (64), Eco FadD (AAA23752), Eco 2-amino-3-ketobutyryl-CoA ligase
(
(
1FC4_A), and Eco crotonobetaine/carnitine CoA ligase CaiC (CAA52113) from E. coli; Hsa Sa protein
BAB68363) from Homo sapiens; Rrh FadD19 (ADP09625) from Rhodococcus rhodochrous; Sam
samR0482 (CAJ88192) from Streptomyces ambofaciens ATCC 23877 (30); Ssp CinT (CBW54660) from
Streptomyces sp. JS360 (31); Tth FACL (Q5SKN9) from Thermus thermophilus Hb8; Tth phenylacetyl-
CoA ligase (YP_004577) from Thermus thermophilus HB27; Mxa FtpD (YP_634753) from Myxococcus
xanthus DK 1622 (17); Bxe benzoyl-CoA ligase (2V7B) from Burkholderia xenovorans Lb400; Bce
phenylacetyl-CoA ligase Paak1 (2Y27_A) from Burkholderia cenocepacia; Awo caffeyl-CoA synthetase
CarB (ADX43862) from Acetobacterium woodii; Bce o-succinylbenzyl-CoA ligase (ADK07438) from
Bacillus cereus biovar anthracis str. CI; Mtu FadD3 (NP_218078.), Mtu FadD5 (NP_214680), Mtu FadD13
(
NP_217605) (58), Mtu FadD15 (NP_216703), Mtu FadD19 (YP_177983), Mtu FadD10 (NP_214613),
Mtu FadD28 (NP_217457) (18), Mtu FadD29 (NP_217466), Mtu FadD30 (NP_214918), Mtu FadD32
NP_218318) (16), Mtu FadD33 (NP_215861), and Mtu FadD34 (YP_177686) from Mycobacterium
(
tuberculosis H37Rv; Ssp RevS (BAK64635) from Streptomyces sp. SN-593, Lma JamA (AAS98774) from
Lyngbya majuscule (11); Kse KSE_65520 (YP_004908267) from Kitasatospora setae KM-6054; Cal
PuwC (AIW82280) from Cylindrospermum alatosporum CCALA 988 (20); Sro DptE (AAX31555) from
Streptomyces roseosporus NRRL 11379 (13); Bgl CayA (AIG53815) from Burkholderia gladioli pv.
agaricicola (21); Ssp salicylyl-CoA ligase (BAC78380) from Streptomyces sp. WA46; Sma benzoyl-CoA
ligase (AAF81733) from Streptomyces maritimus; Bsu YhfL (NP_388908) from Bacillus subtilis subsp.
subtilis str. 168; Lpn FAAL (3KXW) from Legionella pneumophila (64); Set FACL (1PG4) from
Salmonella enterica.
FIGURE 6. Biochemical characterization of RevT. A, SDS-PAGE analysis of RevT using 12.5%
polyacrylamide gel. Lane 1, molecular mass markers; lane 2, His8-tag purified RevT (1 μg). B, size
exclusion chromatography of RevT. Purified protein (0.3 mg) was analyzed on a Superdex 200 column. C,
LC/ESI-MS analysis of RevT reaction products. Synthetic standard of butylmalonyl-CoA (i), synthetic
standard of (E)-2-hexenoyl-CoA (ii), and time-dependent production of butylmalonyl-CoA after 10 min
(
iii), 20 min (iv), and 30 min incubation (v). After heat treatment of RevT at 95C for 5 min, the supernatant
was collected by centrifugation at 15,000 ×g for 30 min at 4C. Heat-treated RevT was incubated for 20
min (vi).
1
3
FIGURE 7. Feeding of labeled fatty acid and C-NMR analysis. A, Biosynthetic scheme of octanoic
13
acid incorporation into 1. B, C-NMR analysis of non-labeled 1 (i, iii) and labeled 1 after feeding of
1
3
13
[
1,2,3,4- C]octanoic acid (ii, iv). C, Biosynthetic scheme of hexanoic acid incorporation into 1. D, C-
1
3
NMR analysis of non-labeled 1 (i) and labeled 1 after feeding of [1- C]hexanoic acid (ii).
FIGURE 8. In vitro enzyme coupling assay. A, Reaction scheme of (E)-2-hexenoic acid into butylmalonyl-
CoA by RevS and RevT. B, LC/ESI-MS chromatogram of butylmalonyl-CoA (i), (E)-2-hexenoic acid (ii),
2
2

Middle chain fatty acyl-CoA ligase involved in reveromycin A biosynthesis
1
h reaction product by RevS and RevT (iii), and 1 h reaction product using RevS and RevT treated at 95C
for 5 min (iv). Peaks corresponding to butylmalonyl-CoA and (E)-2-hexenoic acid were shown at 257 nm
(
blue line) and 240 nm (yellow line), respectively. C, Scheme of RevS and RevT reaction using ACP. D,
Analysis of RevT reaction product by MALDI-TOF/MS. After RevS reaction using (E)-2-nonenoic acid
and holo-ACP, the formation of (E)-2-nonenoyl-ACP was confirmed by MALDI-TOF/MS (i). The RevS
reaction mixture containing (E)-2-nonenoyl-ACP was further reacted in the presence and absence of RevT
(
ii, iii).
2
3

Middle chain fatty acyl-CoA ligase involved in reveromycin A biosynthesis
TABLE 1
Primers used in this study
Primers used for the target gene inactivation by λ-Red system
Target gene
template DNA)
revR
pKD13)
Primers
(
revR-For-P4:
(
5′-GACGAGTGGATCCGCCGGCACTCCGGGATCGTCTCGCGC
ATTCCGGGGATCCGTCGACC-3′
revR-Rev-P1:
5
′-TTCCATCGCCAGCGGGATGGAGGCGGCCGAGGTGTTGCC
TGTAGGCTGGAGCTGCTTC-3′
revS-For-P4:
5′-GCCGACGACGCGGGCGCGAAGACGATCCTCACCACGACC
ATTCCGGGGATCCGTCGACC-3′
revS-Rev-P1:
revS
pKD13)
(
5
′-GTAGAAGTTGCGGCCCTGGTGGATGACCACGTCCTTCAG
TGTAGGCTGGAGCTGCTTC-3′
revT-For-P4:
5′-ACCGTGTGGTCGGCGATGTTCGAGCCCATCTCGACCTTC
ATTCCGGGGATCCGTCGACC-3′
revT-Rev-P1:
revT
pKD13)
(
5
′-TTACACGGACCGGAGCGGGTTGAGCTTCTGCTCACCCAG
TGTAGGCTGGAGCTGCTTC-3′
β-lactamase gene pKD13-aphII-For:
in pWHM3
5′-ATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTT
pKD13)
(
AGAGCGCTTTTGAAGCTCA-3′
pKD13-aphII-Rev:
5
′-TTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGA
GGAACTTCGGAATAGGAACT-3′
Primers used for DNA amplification, including target genes for disruption
Target gene
template DNA)
revR and revS
PCC1FOS-
C11)
Primers
(
ΔrevR/S-HindIII-For:
5′-CCCAAGCTTGCCCGACCGGTACAGCGCGACCAT-3′
ΔrevR/S-HindIII-Rev:
(
3
5
′-CCCAAGCTTCCGAGGCGACCAGGTGCCACAGGT-3′
ΔrevT-HindIII-For:
′-CCCAAGCTTCCAGCGCCACCATCATGGTGAAGA-3′
ΔrevT-HindIII-Rev:
′-CCCAAGCTTGCAGTTCCAACCACGAACCGGAGT-3′
revT
PCC1FOS-
C11)
(
5
3
5
Primers used for heterologous expression in E. coli
Target gene
(
template DNA)
2
4

Middle chain fatty acyl-CoA ligase involved in reveromycin A biosynthesis
revR
PCC1FOS-
C11)
revS
PCC1FOS-
C11)
revT
PCC1FOS-
C11)
acp
revR-NdeI-For:
′-GGAATTCCATATGGCGGGCACGATCGGCACGGCC-3′
revR-XhoI-Rev:
′-CCGCTCGAGTCAGGGCAGGGCGACCACCATCGC-3′
revS-NdeI-For:
′-GGAATTCCATATGGAACTCGCCCTGCCGGCCGAG-3′
revS-XhoI-Rev:
′-CCGCTCGAGTCACGCCTCCCGCGCCGGTGCC-3′
revT-NdeI-For:
′-GGAATTCCATATGGAACCCATGACCGAGGCAGTG-3′
revT-XhoI-Rev:
′-CCGCTCGAGTTACACGGACCGGAGCGGGTTGAG-3′
ACP-NdeI-For:
PCC1FOS-4B1) 5′-GGAATTCCATATGGCCACCAAGGAAGAGATCGTC-3′
ACP-XhoI-Rev:
′-CCGCTCGAGTCAGCCCTGTGCCTTGAGGATGTA-3′
5
(
3
5
5
(
3
5
5
(
3
5
(
5
sfp
sfp-NdeI-For:
(
pUC19-sfp)
5′-CGAATTCCATATGAAGATTTACGGAATTTA-3′
sfp-XhoI-Rev:
5
′-CCGCTCGAGTTATAAAAGCTCTTCGT-3′
Primers used for pTYM19 complementation vector
Target gene
template DNA)
revR
pET28b-revR-
BamHI_mut)
1002 bp)
revS
PCC1FOS-
C11)
1740 bp)
Primers
(
revR-BamHI-For:
5′-CGCGGATCCATGGCGGGCACGATCGGCACG-3′
revR-HindIII-Rev:
5′-CCCAAGCTTTCAGGGCAGGGCGACCACCATC-3′
revS-BamHI-For:
5′-CGCGGATCCATGGAACTCGCCCTGCCGGCC-3′
revS-HindIII-Rev:
5′-CCCAAGCTTTCACGCCTCCCGCGCCGGTGC-3′
revT-BamHI-For:
5′-CGCGGATCCATGGAACCCATGACCGAGGCAGTG-3′
revT-HindIII-Rev:
(
(
(
3
(
revT
PCC1FOS-
C11)
1332 bp)
(
3
(
5′-CCCAAGCTTTTACACGGACCGGAGCGGGTTGAG-3′
List of primer used for Southern blot analysis
Target
gene
Primers
Probe Enzyme
(bp)
Fragment size
(bp)
(
template)
Wild- mutant
type
ΔrevR
pIM-
3608
NcoI
SalI
10808
2889
8520
revR-probe-For:
(
5
′-CCCGAACCGCGAGTAACGCCAGTA-3′
revR-probe-Rev:
′-GATCCTCACCACGACCGCCGTCAA-3′
revS-probe-For:
′-CCCAAGCTTGCCCGACCGGTACAGCG
CGACCAT-3′
ΔrevR)
849
2070
3889
4
509
5
ΔrevS
5180
ApaLI
542
542
1495
2332
6361
(
pIM-
1495
2332
5
ΔrevS)
7279
2
5

Middle chain fatty acyl-CoA ligase involved in reveromycin A biosynthesis
BalI
2615
246
2615
5328
revS-probe-Rev:
′-CCCAAGCTTCCGAGGCGACCAGGTGC
6
5
CACAGGT-3′
revT-probe-For:
ΔrevT
pIM-
ΔrevT)
5399
EcoRI
NotI
4498
3565
(
16321 16321
5
′-CCCAAGCTTCCAGCGCCACCATCA
TGGTGAAGA-3′
revT-probe-Rev:
5109
754
4176
8754
8
5
′-CCCAAGCTTGCAGTTCCAACCACG
AACCGGAGT-3′
Underlines (6–14-bp and 39-bp) show restriction enzyme sites and homologous sequences to the target
DNA region, respectively.
2
6

Middle chain fatty acyl-CoA ligase involved in reveromycin A biosynthesis
TABLE 2
Summary of RevS reaction products
RevS reaction products were analyzed by LC/ESI-MS as described in the Experimental procedures. All
mass spectra were collected in the ESI-negative mode.
Substrates
RevS reaction product
Retention time m/z [M-H]― Structure of product speculated
(
min)
butanoic acida
17.6
21.1
28.1
32.9
38.8
43.2
10.8
13.4
16.4
19.9
24.2
28.9
18.5
23.4
29.3
34.2
39.2
45.2
836
850
864
878
892
906
862
876
890
904
918
932
848
862
876
890
904
918
pentanoic acida
hexanoic acida
heptanoic acida
octanoic acida
nonanoic acida
(
(
(
(
(
(
(
(
(
(
(
(
E)-2-hexenoic acidb
E)-2-heptenoic acidb
E)-2-octenoic acidb
E)-2-nonenoic acidb
E)-2-decenoic acidb
E)-2-undecenoic acidb
E)-3-pentenoic acidc
E)-3-hexenoic acidc
E)-3-heptenoic acidc
E)-3-octenoic acidc
E)-3-nonenoic acidc
E)-3-decenoic acidc
a
b
c
Eluted by a linear gradient from 2–20% acetonitrile for saturated acyl-CoA.
Eluted by a linear gradient from 2–50% acetonitrile for (E)-2-enoyl-CoA.
Eluted by a linear gradient from 2–65% acetonitrile for (E)-3-enoyl-CoA.
2
7

Middle chain fatty acyl-CoA ligase involved in reveromycin A biosynthesis
TABLE 3
Kinetic constants of RevS for fatty acids, ATP, and CoA.
Enzyme
Substrate
K
m
k
cat
k
cat/K
m
－
1
-1
-1
mM
min
mM min
16
RevS
hexanoic acid
heptanoic acid
octanoic acid
0.32 ± 0.07
0.26 ± 0.03
0.32 ± 0.04
0.48 ± 2.44
0.31 ± 0.02
0.59 ± 0.05
0.57 ± 0.05
0.49 ± 0.07
0.48 ± 0.04
0.30 ± 0.04
0.075 ± 0.004
0.95 ± 0.11
0.47 ± 0.06
0.51 ± 0.92
0.71 ± 1.19
0.38 ± 0.03
0.05 ± 0.01
0.039 ± 0.004
0.285
5.2 ± 0.3
18.3 ± 0.5
43.5 ± 1.5
44.3 ± 0.1
19.6 ± 0.3
5.5 ± 0.2
15.1 ± 0.4
35.8 ± 1.5
49.5 ± 1.2
19.3 ± 0.7
6.6 ± 0.1
14.2 ± 0.6
18.2 ± 0.7
41.3 ± 0.9
59.6 ± 1.1
23.6 ± 0.6
39.6 ± 0.8
56.7 ± 2.2
11
70
136
92
nonanoic acid
decanoic acid
64
(
E)-2-hexenoic acid
9.3
27
(
E)-2-heptenoic acid
(
E)-2-octenoic acid
74
(
E)-2-nonenoic acid
103
65
(
E)-2-decenoic acid
E)-2-undecenoic acid
E)-3-hexenoic acid
E)-3-heptenoic acid
(
88
(
15
(
39
(
E)-3-octenoic acid
81
(
E)-3-nonenoic acid
85
(
E)-3-decenoic acid
CoAa
62
861
1456
38
ATPa
Migb
octanoic acid
CoAa
0.045
ATPa
0.036
FadD13c
palmitic acid
CoAd
0.019 ± 0.005
0.13 ± 0.01
0.24 ± 0.05
1.66
87.3
ATPd
At least triplicate assays were performed. The data represents mean ± S.E.M. The kcat/K value was
m
calculated based on the mean value of value K
m
and kcat
.
a
Octanoic acid was used as a substrate.
b
Kinetic parameter reported by Morsczeck et al. (65).
Kinetic parameter reported by Khare et al. (66).
Palmitic acid was used as a substrate (66).
c
d
2
8

Middle chain fatty acyl-CoA ligase involved in reveromycin A biosynthesis
TABLE 4
Comparison of the kinetic constant of RevT and CCR homologs reported
Enzyme
Substrate
K
m
k
cat
k
cat/K
m
－
1
mM－1 min－1
mM
min
RevT
(E)-2-hexenoyl-CoA
0.27 ± 0.01
0.33 ± 0.04
0.12 ± 0.01
0.12 ± 0.01
0.0207 ± 0.0042
0.0044 ± 0.0018
0.95 ± 0.05
0.23 ± 0.01
0.36 ± 0.03
0.078 ± 0.002
28.3 ± 0.5
36.2 ± 1.3
33.9 ± 1.5
41.1 ± 2.0
15.4 ± 0.9
23.1 ± 2.6
82.6 ± 4.4
17.4 ± 1.1
0.218 ± 0.011
0.112 ± 0.006
104
(
E)-2-octenoyl-CoA
NADPHa
109
282
NADPHb
342
SalGc
CinFd
SpnEe
crotonyl-CoA
744
chlorocrotonyl-CoA
crotonyl-CoA
5250
86.6
(
E)-2-octenoyl-CoA
crotonyl-CoA
75.7
0.614
1.43
cinnamoyl-CoA
At least triplicate assays were performed. The data represents mean ± S.E.M. The kcat/K
calculated based on the mean value of value K
m
value was
m
and kcat.
a
(
E)-2-hexenoyl-CoA was used as a substrate.
b
c
d
e
(
E)-2-octenoyl-CoA was used as a substrate.
Kinetic parameter reported by Eustáquio et al. (27).
Kinetic parameter reported by Quade et al. (31).
Kinetic parameter reported by Chang et al. (67).
2
9

Fig. 1
(I)
1
A
R
(III)
OH
O
O
O
2
R
O
H
O17
COOH
H
O
H
1
NH
O
O
18
O
1
HO
NH
O
R
H
N
O
COOH
OH
HOOC
O
H
O
O
O
OH
2
R
O
O
Cl
O
cinnabaramides
A : R =OH, R =H
B : R =OH, R =OH
C: R =H, R =H
H
O
reveromycins (RM)
RM-A (1) : R =H, R =H
RM-C (2): R =H, R =CH
RM-D (3): R =CH
1
2
HO
1
2
salinosporamide A
1
2
1
2
3
1
2
1
2
FK506
3
, R =H
1
2
RM-E (4) : R =H, R =CH
2
CH
3
(IV)
OH
OH
OH OH
(II)
O
R =
stambomycin A
1"
O
2" R
OH
R
OH
HO
OH
R =
stambomycin B
R =
stambomycin C
R =
stambomycin D
O
H
N
O
O
O
O
germicidin F : R=H
germicidin G : R=CH
HN
3
HO
HO
HO
HO
HO
H
O
O
O
HO
HO
O
OH
OH
O
N
O
HO
O
O
OH
O
OH
OH OH
OH
ansalactam A
divergolide C
B
reveromycins
revT homolog gene
revS homolog gene
revR homolog gene
revT
revS
revR
unknown gene cluster
from Kitasatospora setae
(I)
KSE_65510 KSE_65520
KSE_65530
cinL
cinnabaramides
cinT
anlE
cinS
cinF
propionyl-CoA carboxylase
β-subunit homolog gene
ansalactams
anlF anlG
divS divT
(II)
germicidins and divergolides
divR
salinosporamides
FK506
salT salS
tcsA
salQ
tcsC
salN salM
salL
salG
salH
(III)
tcsB
tcsD
stambomycins
(IV)
1
kb
samR0483
samR0482
ACP
ACP
ACP
ACP
(
)
( )
( )
( )
C
OH
O
S
R
CoA
S
CoA
S
CoA
SH
O
O
O
CoA
CO
2
COOH
fatty acid
degradation
1
2
+
ATP AMP + PPi
NADPH NADP
R
R
RevT
RevS
β-oxidation
transacylation
S
CoA
O
COOH
KASIII
S
ACP
S
ACP
S
ACP
S
O
O
O
O
O
O
S
CoA
OH
O
COOH
KASIII (RevR)
FabG, FabZ
R
3
de novo fatty acid
biosynthesis
FabG, FabZ
KASIII
ACP
S
S
CoA
ACP
S
ACP
S
O
COOH
S
R
4
O
O
O
O
O
O
O
OH
R
FabG, FabZ, FabI,
FabB/F, FabH
KASIII (FabH)
R
2-alkylmalonyl-CoA (ACP)