Characterization of 2-octenoyl-CoA carboxylase/reductase utilizing pteB from Streptomyce avermitilis

Article abstract of DOI:10.1271/bbb.110003

The filipin biosynthetic gene cluster of Streptomyces avermitilis contains pteB, a homolog of crotonyl-CoA carboxylase/reductase. PteB was predicted to be 2-octenoyl-CoA carboxylase/reductase, supplying hexylmalonyl-CoA to filipin biosynthesis. Recombinant PteB displayed selective reductase activity toward 2-octenoyl-CoA while generating a broad range of alkylmalonyl-CoAs in the presence of bicarbonate.

Full text of DOI:10.1271/bbb.110003

Biosci. Biotechnol. Biochem., 75 (6), 1191–1193, 2011
Note
Characterization of 2-Octenoyl-CoA Carboxylase/Reductase Utilizing pteB
from Streptomyce avermitilis
1
1
1
1
Hye-Gyeong YOO, So-Yeon KWON, Suji KIM, Suman KARKI,
2
1;y
Zee-Yong PARK, and Hyung-Jin KWON
1
Department of Biological Science, Division of Bioscience and Bioinformatics, Myongji University,
Yongin 449-728, Republic of Korea
2
Department of Life Sciences, Gwangju Institute of Science and Technology, Gwangju 500-712, Republic of Korea
Received January 5, 2011; Accepted March 16, 2011; Online Publication, June 13, 2011
[doi:10.1271/bbb.110003]
The filipin biosynthetic gene cluster of Streptomyces
production of chloroethylmalonyl-CoA. Strop 3612 enc-
odes a CCR with housekeeping properties that might
act as the major biosynthetic machinery in producing
ethylmalonyl-CoA in S. tropica.
avermitilis contains pteB, a homolog of crotonyl-CoA
carboxylase/reductase. PteB was predicted to be 2-
octenoyl-CoA carboxylase/reductase, supplying hexyl-
malonyl-CoA to filipin biosynthesis. Recombinant PteB
displayed selective reductase activity toward 2-octenoyl-
CoA while generating a broad range of alkylmalonyl-
CoAs in the presence of bicarbonate.
The polyketide biosynthetic gene cluster of filipin in
Streptomyces avermilitis contains a homolog of the CCR
7
,8)
gene, pteB. The chemical structure of filipin suggests
that the last chain-extension step incorporates hexylma-
lonyl-CoA. This is the basis of our hypothesis that PteB
is 2-octenoyl-CoA carboxylase/reductase (Fig. 1AB).
Notably, the S. avermitilis genome also harbors at least
two other CCR encoding genes (one previously identi-
Key words: 2-octenoyl-CoA
carboxylase/reductase;
crotonyl-CoA carboxylase/reductase; Strep-
tomyces avermitilis; polyketide synthase;
filipin biosynthesis
7
)
fied in the oligomycin biosynthetic gene cluster).
We found that recombinant PteB selectively reduced
2-octenonyl-CoA in the absence of bicarbonate while
inclusion of bicarbonate drove PteB to catalyze reduc-
tive carboxylation toward crotonyl-, 2-hexenoyl-, 2-
octenoyl-, and 2-decenoyl-CoA.
Crotonyl-CoA reductase (CCR) of Rhodobacter
sphaeroides was recently redefined as crotonyl-CoA
carboxylase/reductase, supplying ethylmalonyl-CoA in
1
,2)
C5-dicarboxylic acid biosynthesis. It was immediately
recognized that CCR might play a role in supplying
C4-extension unit, in the form of ethylmalonyl-CoA,
Polymerase chain reaction (PCR) was used to amplify
pteB from the total DNA of S. avermitilis DNA utilizing
1
)
0
in polyketide biosynthesis. Many bacterial polyketide
biosynthetic gene clusters harbor a homolog of ccr, and
the cognate polyketide structure incorporates the C4-
extension unit. This is exemplified by the FK520
the following primers: 5 -ATTCATATGACGAAAGC-
0
0
TCTCTACGA-3 and 5 -TTCAAGCTTTCAGGCGGC-
0
GGCGAGTTCGA-3 (engineered NdeI and HindIII sites
underlined). PCR-amplified pteB was sub-cloned in
pET28a, and protein synthesis was induced in E. coli
BL21 (DE3) with 1 mM isopropyl ꢀ-D-1-thiogalactopy-
3
)
biosynthetic gene cluster. In the catalysis of modular
polyketide synthase (PKS), an acyltransferase domain
ꢀ
(
AT) selects a malonyl-CoA or an alkylmalonyl-CoA
ranoside at 14 C overnight (Fig. 1C). 2-Alkenoyl-CoA
precursor and charges the neighboring acyl-carrier
protein (ACP) with it. ꢀ-Ketoacyl-ACP synthase incor-
porates the selected precursor unit into the growing
polyketide chain through decarboxylative Claisen con-
densation. The precursor selection step is pivotal in
generating the structural diversity of polyketides. Ex-
tensive research efforts have hence been directed toward
understanding the biosynthesis of atypical malonyl-CoA
derivatives and the biochemical mechanisms guiding
their selective incorporation into growing polyketide
chains.4
substrates, except for crotonyl-CoA, were generated by
reaction of the cognate alkanoyl-CoA with acyl-CoA
oxidase (ACOD, Wako Pure Chemical Industries,
Osaka, Japan). Crotonyl-CoA, hexanoyl-CoA, octanoyl-
CoA, and decanoyl-CoA were purchased from Sigma-
Aldrich (St. Louis, MO). One mM alkanoyl-CoA was
incubated with 0.1 U ACOD in 100 mL of 100 mM
potassium bicarbonate and 50 mM Tris–HCl (pH 7.5)
ꢀ
for 20 min at 37 C. The reaction mixture was boiled for
2 min, and transferred to an ice bath, and then 2 mM
reduced nicotinamide adenine dinucleotide phosphate
(NADPH) and 3 mM PteB were added. The final
concentration of potassium bicarbonate was 80 mM in
the PteB reaction. Crotonyl-CoA was used in the
reaction without ACOD treatment. High-performance
liquid chromatography (HPLC) indicated that ACOD
successfully generated 2-alkenyl-CoA, as verified by
negative ESI-MS analysis utilizing the detection of
)
Biochemical characterization of the CCR route in
polyketide biosynthesis was first reported for the
5
,6)
salinosporamide biosynthesis of Salinospora tropica.
SalG catalyzes the formation of ethylmalonyl-CoA,
propylmalonyl-CoA, and chloroethylmalonyl-CoA to
support the production of salinosporamide derivatives.
The metabolic significance of SalG might lie in its
y
To whom correspondence should be addressed. Tel: +82-31-330-6470; Fax: +82-31-336-0870; E-mail: hjink@mju.ac.kr

1192
H.-G. YOO et al.
þ ꢁ
½
M ꢁ H ꢂ at m=z 862.3, 890.3, and 918.2 for 2-
hexenoyl-, 2-octenoyl-, and 2-decenoyl-CoA respectively
Fig. 2). Alkanoyl-CoA substrate was left in the various
A
(
ACOD reactions, and complete conversion to 2-alkenoyl-
CoA was not achieved because the ACOD reaction
generates by-products when incubated for longer than
20 min.
The various PteB reactions generated new peaks with
B
C
consumption of 2-alkenoyl-CoAs (Fig. 2). The samples
corresponding to the new peaks were collected and
analyzed by mass spectrometry. Electrospray ionization-
mass spectrometry (ESI-MS) in negative mode verified
the identities of the alkylmalonyl-CoAs by accessing the
þ 2ꢁ
detection of ½M ꢁ 2H ꢂ at m=z 439.7, 453.6, 467.7,
and 481.6 for ethyl, butyl, hexyl, and octylmalonyl-CoA
respectively. The conversion of crotonyl-CoA into
ethylmalonyl-CoA was not completed, and ESI-MS
verified the identity of the crotonyl-CoA that was left in
the PteB reaction (Fig. 2A). Analyses of these HPLC
results indicated that recombinant PteB displays broad
substrate specificity for in vitro reductive carboxylation.
Notably, the level of alkanoyl-CoA was slightly en-
hanced in the PteB reaction, especially with 2-decenoyl-
CoA. This enhanced level indicates that some 2-alkenyl-
CoAs are converted into the cognate alkanoyl-CoAs
through reduction decoupled with carboxylation
Fig. 1. Filipin III Structure (A), Reaction Scheme for 2-Alkenoyl-
CoA Carboxylase/Reductase (B), and Purification of PteB (C).
C, Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of
(Fig. 1B). When the bicarbonate concentration was
reduced to 1 mM, the carboxylation-decoupled reduction
appeared enhanced, whereas overall reductive conver-
sion was diminished (data not shown).
The substrate specificity of PteB was further exam-
ined in a UV-Vis spectrophotometer by monitoring of
NADPH oxidation. This experiment showed that PteB
selectively reduced 2-octenoyl-CoA in the absence of
bicarbonate (Fig. 3A). Marginal NADPH oxidation was
observed with 2-hexenoyl-CoA. When 80mM bicarbonate
was included, PteB displayed broad substrate specificity,
6
ꢃ histidine tagged recombinant PteB purified by nickel affinity
chromatography on Ni-NTA agarose. Lane M, molecular weight
protein markers (Amersham; 225, 150, 102, 76, 52, 38, 31, 24, 17,
and 12 kDa top to bottom respectively with 52- and 38-kDa proteins
indicated); lane 1, total proteins; lane 2, flow-through; lane 3,
elution with 20 mM imidazole; lanes 4–7, elution with 250 mM
imidazole.
C
D
A
B
Fig. 2. HPLC Analysis of PteB Reactions with 2-Alkenoyl-CoAs.
PteB reaction with crotonyl-CoA (A) and ACOD reactions (B–D) are shown by black solid lines; hexanoyl-CoA (B), octanoyl-CoA (C), and
decanoyl-CoA (D). ACOD reaction samples and authentic alkanoyl-CoAs are shown by gray solid lines and black dotted lines respectively.
Alkylmalonyl-CoAs, 2-alkenoyl-CoAs, and alkanoyl-CoAs are indicated by solid circles, hollow circles, and arrows respectively. The reactions
were monitored by HPLC on a Gemini C-18 column (150 ꢃ 2 mm, 3.0 mm; Phenomenex, Torrance, CA) at 260 nm. The mobile phase consisted
of 5 mM ammonium acetate and 5% methanol in water (A) and methanol (B). The flow rate was maintained at 0.5 mL/min. The column was
programmed to run in a timed gradient fashion: 100% A for 5 min, transitioning from 100% A to 10% A over the next 25 min, then maintained at
10% A for 10 min. The sample injection volume was 25 mL.

2
-Octenoyl-CoA Carboxylase/Reductase in Filipin Biosynthesis
1193
2
-octenoyl-CoA carboxylase/reductase (TgaD).11) Phy-
A
logenetic analysis indicated that TugD and PteB con-
stitute separate clades (data not shown). It is likely that
tugD and pteB evolved independently in myxobacteria
and actinobacteria respectively. All known thuggacins
of S. cellulosun origin possess a hexyl group at the
ꢁ
-position of the macrolactone ring. An identical
substituent is also present in filipins. Thus it can be
hypothesized that the cognate AT of filipin and thuggacin
PKS selectively incorporate hexylmalonyl-CoA. It is
speculated that PteB retains relaxed substrate specificity
due to pteB evolution from the ccr gene.
In conclusion, 2-octenoxyl-CoA carboxylase/reductase
was characterized in filipin biosynthesis. PteB can be
employed in combinatorial biosynthesis to generate
polyketide structures from various alkyl precursors if the
biosynthetic reaction involves an AT with relaxed
substrate specificity, as seen for salinosporamide and
B
6
,9)
FK506.
Acknowledgment
This work was supported by the Korea Research
Foundation Grant funded by the Korean Government
(MOEHRD, Basic Research Promotion Fund) (2009-
0074283) and a grant from the Next-Generation
Fig. 3. Spectrophotometric Analysis of PteB Reactions with 2-
Alkenoyl-CoAs.
PteB reactions with crotonyl-CoA (solid triangles), ACOD
reaction of hexanoyl-CoA (hollow circles), octanoyl-CoA (solid
circles), and decanoyl-CoA (hollow triangles) were performed in
BioGreen 21 Program (PJ007987), Rural Development
Administration, Republic of Korea.
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4
,5)
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¨