Fatty Acid Biosynthesis in Bacilli
2113
supported by their resistance to the antibiotic thiolac-
tomycin, which selectively inhibits dissociated Type
II, but not Type I, FAS.16) Thus the studies on KAS
entities in Bacilli may unveil the novel and unique
features of this enzyme, and may address the ques-
tions raised by this study.
protein may alternatively agree in part with the previ-
ous observation that the reconstituted polyketide
synthase (PKS) from Streptomyces coelicolor pro-
duced polyketides without MAT when holo ACP is
present in excess. The polyketides are synthesized by
a similar mechanism that resembles fatty acid biosyn-
thesis. In that case, the PKS-ACP catalyses sel-
fmalonylation, and provides malonyl-ACP for the
successive condensing reaction.18–20) As is the case of
PKS, it also may be possible that the ACP of B. sub-
tilis undergoes selfmalonylation, and may serve as a
chain extension unit of fatty acid biosynthesis. To
address this question, we studied the selfmalonyla-
tion of B. subtilis ACP under the same conditions as
the MAT assay, but in the absence of a MAT enzyme
source. The puriˆed B. subtilis ACP alone, however,
showed no selfmalonylation in this study (data not
shown), and this may negate the possibility above
that the ACP selfmalonylation was responsible for
the FabD independent BCFA biosynthesis.
We herein ˆrst demonstrated that the FabD pro-
tein is not essential for in vitro BCFA biosynthesis by
the crude FASs of B. subtilis. We applied the im-
munoprecipitation method to remove FabD protein
from the crude BCFA synthetase. The antibody pre-
pared in this study speciˆcally recognized the FabD
protein in the crude FAS (Figs. 4 and 6), and was
found to be eŠective to remove FabD protein from
the crude system. By use of a similar method, our
previous study has demonstrated the requirement of
BCKA decarboxylase for the BCFA biosynthesis,
and conˆrmed the usefulness of the immunoprecipi-
tation method.15) The supernatant after im-
munoprecipitation in this study thus speciˆcally
lacked FabD protein retaining with activities of all
other members of FASs. However, the removal of
FabD did not necessarily eliminate the in vitro BCFA
biosynthetic activity from the crude FAS, but only
Although this study ˆrst demonstrated that BCFA
biosynthesis by crude FAS did not necessarily require
FabD protein, the current in vitro results should be
reasonably related to in vivo experiments. One
experimental approach for this purpose is to study
BCFA biosynthesis in an FabD-deˆcient mutant. The
FabD-deˆcient mutant of E. coli was an auxotroph
requiring both saturated and unsaturated fatty acid.1)
The authors hope that this paper triggers some
studies to clarify the metabolic link between fatty
acid biosynthesis and other pathways including
reduced the level to 50–60
z
for the case of BCACs,
and to 80 for KMV primer. This ˆnding clearly
z
indicates that the FabD in the crude system is not
essential for the BCFA biosynthesis.
The point to mention is that there may be an
alternate enzyme source that can catalyze the MAT
reaction. We attempted to identify another enzyme
source in the crude FAS, and detected alternate MAT
activity apart from FabD protein (Fig. 7). Thus, the
observation that the removal of FabD from the crude
FAS did not eliminate the MAT activity in the crude
enzyme may be explained by the presence of another
enzyme source for FabD. A search of the B. subtilis
genome database found three MAT homologues de-
scribed as involved in polyketide synthesis: PksC,
PksE, and PksD with the similarity of 56, 54, and
polyketide synthesis in B. subtilis
.
References
1) Magnuson, K., Jackowski, S., Rock, C. O., and
Cronan, J. E., Regulation of fatty acid biosynthesis
in Escherichia coli. Microbiol. Rev., 57, 522–542
(1993).
2) Rock, C. O., and Cronan, J. E., Escherichia coli as a
model for the regulation of dissociable (type II) fatty
acid biosynthesis. Biochim. Biopys. Acta, 1302, 1–16
(1996).
3) Kaneda, T., Iso- and anteiso-fatty scids in bacteria:
biosynthesis, function and taxonomic signiˆcance.
Microbiol. Rev., 55, 288–302 (1991).
40
z
to FabD, respectively. The polyketide synthase
(PKS) and FAS are related not only in their mechan-
ism of carbon chain extension but also in their prima-
ry sequences. It thus appeared hold true that these
two systems evolved from a common origin after ear-
ly gene duplication. As the many biochemical steps
of FAS and PKS are similar, it seems quite reasona-
ble that these two system share common enzymatic
activities up to the point at which the pathways
diverge. In this context, it is noteworthy that FabD
has been implicated as functioning to charge the
ACP subunit of FAS and PKS prior to the condensa-
tion reaction.17) Thus the gene product of MAT
homologues in the crude extract may be a potential
alternate enzyme source, and may compensate the
deˆciency of the FabD activity for BCFA biosynthe-
sis.
4) Butterworth, P. H. W., and Bloch, K., Comperative
aspects of fatty acid synthesis in Bacillus subtilis and
Escherichia coli
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5) Choi, K.-H., Heath, R. J., and Rock, C. O.,
b
-Keto-
acyl carrier protein synthase III (FabH) is a determin-
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6) Morbidoni, H. R., Mendoza, D., and Cronan, J. E.,
Bacillus subtilis acyl carrier protein is encoded in a
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7) Kaneda, T., Biosynthesis of branched long-chain
fatty acids from the related short-chain
a-keto acid
The independence of BCFA biosynthesis on FabD