Download Partial purification of fatty acid synthetase from Streptomyces

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603rd MEETING, LIVERPOOL
Partial purification of fatty acid synthetase from Streptomyces coelicolor
mg of protein) was generally eluted in fractions Corresponding to
mol.wt. 180000. A similar purification was obtained after
sucrose-gradient centrifugation to give material with s20,w
8 S.
Thus the synthetase migrated as a single species during these
Filamentous bacteria of the genus Streptomyces are extremely procedures (with a consistent recovery of over 80%), and its
versatile in making antibiotics, many of which are phenolic activity presumably resides in multifunctional polypeptides of
derivatives formed by reaction of a primer with malonyl-CoA. relatively high molecular weight; it therefore belongs to the type
This pathway leading to the synthesis of the initial aromatic I category.
The properties of the polypeptides identified, however,
product is very similar to the sequence established for fatty acid
synthesis. Presumably, therefore, the two synthetases concerned correspond more closely to those associated with the vertebrate
synthetase (rather than those found in M. smegmatis, with
might show similar properties.
N o information is currently available concerning the nature of mol.wt. 290000), which exists as a homodimer in its native form
the fatty acid or aromatic synthetase in Streptomyces spp., with mol.wt.480000 (Stoops & Wakil, 1981), but is cold-labile
although it might be expected that the former would resemble and dissociates at 0°C (Muesing et al., 1975) into monomeric
the type I complex of mol.wt. 2 x lo6 (comprising multi- species with mol.wt. approx. 220000 (Stoops & Wakil, 1981).
functional polypeptides) found in Mycobacterium smegmatis Accordingly, enzyme preparations from S. coelicolor were
(Wood et al., 1978), rather than the loose aggregate of enzymes tested for possible increased activity after preincubation at
(type I1 system) present in Escherichia coli. We therefore temperatures ranging from 0 to 30OC; activity was enhanced
decided to examine the properties of fatty acid synthetase in considerably above 10°C, as shown for example in Table 1.
Streptomyces coelicolor, an organism which produces the Moreover, when the gel filtration and sucrose-gradient-cenmalonate-derived actinorhodin, a pigmented antibiotic (Wright trifugation procedures were repeated at the higher temperatures,
& Hopwood, 1976), and one which has been extensively a biphasic profile of activity was noted, corresponding to protein
characterized genetically (Wright & Hopwood, 1976; Rudd & with mol.wt. approx. 350000 and 180000 and an s20,wvalue of
13 S and 8 S respectively, confirming the dimeric nature of the
Hopwood, 1980).
Cells were grown under submerged conditions in the complete enzyme under these conditions. Species of synthetase with
medium described by Hopwood (1 967) to mid-exponential higher molecular weight were never detected.
The heat-stable factor increased synthetase activity over
phase, harvested by filtration and disrupted in 0.2 M-phosphate,
pH 7.0 (containing 1mM-phenylmethanesulphonyl fluoride), by 20-fold, but could not be replaced by 2,6-dimethylated cyclotwo passages through a French Press. Synthetase was initially dextrins (Machida et al., 1973), which only gave a maximum of
precipitated by 50-85% saturation with (NH,),SO, (with 1.S-fold stimulation, unlike the situation found in M. smegmatis.
minimal loss of activity), and assayed routinely by measuring Bovine serum albumin was also ineffective.
Thus the Evidence indicates that S. coelicolor (and prethe incorporation of [ 2-14C]malonyl-CoA into long-chain fatty
acids (produced as their CoA thioesters). The assay system sumably other Streptomyces species) contains a novel fatty acid
contained isobutyryl-CoA as primer, [2-*4Clmalonyl-CoA, synthetase comprising two multifunctional polypeptides, with
NADPH, NADH, FMN, dithiothreitol (see Wood et al., 1978), overall mol.wt. approx. 350000, which is dimeric and coldand a heat-stable factor obtained after 50-90% (v/v)-acetone labile. It resembles the vertebrate enzyme in these characterprecipitation of the heat-treated crude extract. The phos- istics and is distinct from that isolated from M. smegmatis and
pholipids in this organism contain fatty acids of predominantly related Actinomycetes. However, it does form acyl-CoA
products with the aid of a heat-stable activator. It may be
the iso- and anteiso-types.
Partial purification of fatty acid synthetase was achieved after speculated that the aromatic synthetase in Streptomyces spp.
gel filtration through Sephadex G-200 when activity (lornunits/ comprises a similar overall structure, which is distinct from fatty
acid synthetase (for example, in its reductive capacity), but
related to it genetically.
STEPHEN FLATMAN and NEVILLE M. PACKTER
Department of Biochemistry, University of Leeds, Lee&
LS2 9JT, U.K.
Table 1. Effect of preincubation at various temperatures on
fatty acid synthetase activity
Synthetase (0.1 ml from a sucrose-gradient fraction) was
preincubated at various temperatures for 20min and then added
to 0.9ml of the assay system at 3OOC. The reaction was stopped
after further incubation at 3OoC for 5 or 15min.
Total radioactivity incorpor- Incorporation relative
Preincubation ated into fatty acids (c.p.m.)
to O°C value
,-*-(
temperature
5min
15-5min
5min
15min 15-5min
("C)
0
10
18
25
Vol. 11
I70
190
220
600
800
1050
1540
2930
630
860
1320
2330
We thank the S.E.R.C. and the John Innes Institute, Norwich, for a
C.A.S.E. award to S. F.
Hopwood, D. A. (1967)Bacteriol. Rev. 31,373-403
Machida, Y.,Bergeron, R., Flick, P. & Bloch, K. (1973)J. Bid. Chem.
248,6246-6241
Muesing, R. A., Lornitzo, F. A., Kumar, S. & Porter, J. W.(1975)J.
Biol. Chem. 250,1814-1823
Rudd, B. A. M. & Hopwood, D. A. (1980)J. Gen. Microbiol. 119,
333-340
1
.o
1 .o
Stoops, J. K. & Wakil, S.J. (1981)J.Biol. Chem. 256,5128-5133
Wood, W. I., Peterson, D. 0. & Bloch, K. (1978)J. Biol. Chem. 253,
1.1
1.3
1.4
2.1
3.7
Wright, L. F. & Hopwood, D. A. (1976) J . Gen. Microbiol. 96,
3.5
2650-2656
289-291