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The resulting E. coli cell pellets were suspended in lysis buffer A
(50 mm sodium phosphate buffer pH 7.2, 300 mm NaCl, 5 mm 2-
mercaptoethanol, 10% glycerol, 0.05% (v/v) Tween-20) with 10 mm
imidazole and lysozyme (1 mgmLÀ1). The resulting cell suspension
was incubated on ice for 30 min, then the cell lysate was cleared
by centrifugation at 16000 g for 20 min. The crude cell extract was
loaded onto a Ni-NTA resin column. The N-terminal polyhistidine-
tagged protein was eluted by using buffer A with imidazole
(300 mm). Fractions containing pure protein were pooled, ex-
changed with 50 mm sodium phosphate buffer pH 7.2, and stored
in 20% glycerol at À808C.
the SCO1814 enoyl-ACP reductase homologue from S. coelicol-
or, and purified all proteins. We carried out the first kinetic
analyses of these streptomycetes enzymes and demonstrated
that all three FabGs homologues have 3-ketoacyl-ACP reduc-
tase activity, with the FabI homologue having enoyl-ACP re-
ductase activity. The activities of the SCO1815-encoded FabG
and the SCO1814 FabI provide compelling support for a role
of these enzymes in providing both fatty acids for primary
metabolism and dodecanoic acid for undecylprodiginine bio-
synthesis (Scheme 1). Thus, the data now provide experimental
evidence to support the role of FabG and FabI, in addition to
FabD, in both processes and suggests they might exert no
control over the products. In contrast, the condensing en-
zymes (FabH and RedP) are the key controlling factors.[14]
General procedures for synthesis of (E)-S-(2-acetamidoethyl) alk-
2-enethioate (enoyl-NAC thioester) substrates for FabI assays:
Straight- and branched-chain enoyl-NAC compounds, ranging from
C4 to C10 in alkyl chain length, were synthesized according to gen-
eral Scheme 2. Reactions were monitored by TLC, and all com-
pounds were characterized by 1H NMR spectroscopy.
Synthesis of alkanal 1: Dimethyl sulfoxide (6.2 mL, 88 mmol) was
added dropwise to a stirred solution of oxalyl chloride (3.80 mL,
44 mmol) in anhydrous CH2Cl2 (100 mL) at À788C under argon.
After 30 min, a solution of the starting alcohol (3.0 g, 29 mmol)
was added dropwise, and the reaction mixture was stirred at
À788C for 1.5 h. Triethyl amine (20.45 mL, 147 mmol) was then
added dropwise, and the reaction mixture was allowed to warm to
room temperature. Saturated aqueous NH4Cl (30 mL) was added,
and the organic layer was separated. The resulting aqueous phase
was extracted with CH2Cl2 (3ꢁ40 mL), and the combined organic
layers were dried over anhydrous sodium sulfate (Na2SO4) and con-
centrated in vacuo to give aldehyde 1.
Experimental Section
Materials: b-NADH and b-NADPH were from Research Products In-
ternational Corp. All other chemicals, including unlabeled CoA de-
rivatives, were from Sigma. Cosmid 3F7 and 4A7, containing S. coe-
licolor genomic DNA, were kindly provided by the John Innes Insti-
tute. FabC, RedQ, and E. coli ACP were sourced as described previ-
ously.[1,30,31] Preparation of malonyl-ACPs and crotonoyl-ACPs was
carried out as described previously.[14]
S. coelicolor and E. coli fabG: The genes encoding SCO1815,
SCO1345, and SCO1346 (fabG) were amplified from the appropriate
S. coelicolor cosmids by PCR by using the primers 5’-CATATG
AGCCGC TCGGTT CTC-3’ and 5’-GGATCC TCAGTG ACCCAT TCCCAG
TCC-3’ (SCO1815); 5’-CATATG ACTGAA CTGCCC GAGCCC TCC-3’
and 5’-GGATCC TCAGGC CACGCC GGCGTT C-3’ (SCO1345); and 5’-
CATATG TCCACC ACTGAG CAGCG-3’ and 5’-GGATCC CTAGTC
GAGCGG TCCGCC-3’ (SCO1346). The E. coli fabG was amplified
from E. coli genomic DNA by PCR by using 5’-CATATG AATTTT
GAAGGA AAAATC GCACTG-3’ and 5’-GGATCC TCAGAC CATGTA
CATCCC GCCG-3’ (restriction sites underlined). The amplified inserts
were digested with NdeI and BamHI and cloned into the pET15b
vector to provide the expression plasmid for SCO1815 (pRS1),
SCO1345 (pRS2), and SCO1346 (pRS3), as well as E. coli fabG
(pRSE1).
Synthesis of (E)-methyl alk-2-enoate (2): Methyl diethylphospho-
noacetate (5.86 g, 27 mmol) was added dropwise to a stirred solu-
tion of the sodium hydride (0.66 g, 27 mmol) in 1.2-dimethoxy-
ethane (15 mL) at 08C under argon atmosphere and stirred for
30 min. After dropwise addition of the aldehyde (1; 2.0 g,
23 mmol), the reaction mixture was allowed to warm to room tem-
perature and stirred for 3 h. The reaction mixture was quenched
with cold water and extracted with EtOAc (3ꢁ15 mL). The com-
bined organic layers were washed with brine (20 mL), dried over
Na2SO4, and concentrated in vacuo. The crude product was purified
by column chromatography (EtOAc/hexane, 7:93) to yield the
desired ester 2.
Synthesis of (E)-alk-2-enoic acid (3): LiOH (1.0 g, 42.2 mmol) was
added to a stirred solution of ester 2 (2.0 g, 14 mmol) in tetrahy-
drofuran/H2O (1:1, 40 mL), and the mixture was heated at reflux.
After 2.5 h, the reaction mixture was cooled to 08C and acidified
with 1n HCl. The layers were separated, and the aqueous phase
was extracted with EtOAc (3ꢁ30 mL). The combined organic layers
were washed with brine, dried over Na2SO4, and concentrated in
vacuo to give the corresponding carboxylic acid (3).
S. coelicolor and E. coli fabI: The SCO1814 gene (encoding FabI)
was amplified from the appropriate S. coelicolor cosmid by using
the forward primer 5’-CATATG AGCGGA ATTCTC GAGGGC AAG-3’
(introducing a NdeI restriction site) and a reverse primer 5’-
GGATCC TCAGGC GCCGAT GGCGTG C-3’ (introducing a BamHI re-
striction site). The E. coli fabI was amplified from E. coli genomic
DNA by using the forward primer 5’-CATATG GGTTTT CTTTCC
GGTAAG-3’ (introducing a NdeI restriction site) and reverse primer
5’-GGATCC TTATTT CAGTTC GAGTTC G-3’ (introducing a BamHI re-
striction site). The resulting PCR products were digested with NdeI
and BamHI and ligated into the corresponding sites in pET15b to
provide the S. coelicolor fabI expression plasmid, pRS4, and the
E. coli fabI expression plasmid, pRSE2.
Synthesis of (E)-S-(2-acetamidoethyl) alk-2-enethioate (4): N-
Acetyl cysteamine (0.94 mL, 8.98 mmol) was added to a stirred so-
lution of carboxylic acid 3 (1.0 g, 7.81 mmol) in anhydrous CH2Cl2
(20 mL) at 08C under nitrogen atmosphere, followed by 4-(N,N-di-
methylamino)pyridine (DMAP; 0.24 mg, 1.95 mmol), and N-(3-dime-
thylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDCl; 1.5 g,
7.81 mmol). The reaction mixture was allowed to warm to room
temperature and stirred overnight. The reaction was quenched
with saturated aqueous NH4Cl (15 mL) and extracted with CH2Cl2
(3ꢁ20 mL). The combined organic layers were dried over Na2SO4,
concentrated in vacuo, and the residue was purified by column
chromatography (hexane/EtOAc, 40:60) to yield desired product 4.
Protein expression and purification: All plasmids were transform
into E. coli BL21(DE3) cells. The resulting transformants were grown
at 378C in LB medium containing ampicillin (100 mgmLÀ1) until
A600 =0.6 was reached, induced with isopropyl-b-d-thiogalactopyra-
noside (0.1 mm), and incubated for 3 h at 378C. Cells were harvest-
ed by centrifugation for 10 min at 12000g and 48C and stored at
À808C.
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Singh, Renu
