Partial purification of fatty acid synthetase from <i>Streptomyces coelicolor</i>

Flatman, S.; Packter, Neville M.

doi:10.1042/bst0110597

Cited by 13 publications

(11 citation statements)

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“…In all of these cases the bacteria appear to compensate for the loss of the branched-chain anteiso-C 15 acids, which are important for reg- (15), by producing unsaturated straight-chain fatty acids. The thiolactomycin-dependent decrease in the levels of isopalmitate and other branched-chain fatty acids was matched by an analogous decrease in the levels of labeled isopalmitate obtained when D 6 -isobutyrate was included in the fermentation broth. This in vitro data indicates that thiolactomycin inhibits branched-chain fatty acid biosynthesis from isobutyrylCoA (in agreement with in vitro data).…”

Section: Discussionmentioning

confidence: 77%

“…Initial reports of fatty acid biosynthesis in Streptomyces coelicolor and Saccharopolyspora erythraea (formerly Streptomyces erythraeus) indicated a type I FAS (6,28). More recent enzymatic and genetic analyses of fatty acid biosynthesis in S. coelicolor, Streptomyces glaucescens, S. collinus, and Saccharopolyspora erythraea have indicated that these systems have a type II FAS (23,24,32,38).…”

Section: Discussionmentioning

confidence: 99%

“…Initial studies suggested that fatty acids in streptomycetes are biosynthesized by a type I fatty acid synthase (FAS) (6,28). Such synthases are large, nondissociable, multifunctional complexes which are generally found in yeast and mammalian systems.…”

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confidence: 99%

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In vivo and in vitro effects of thiolactomycin on fatty acid biosynthesis in Streptomyces collinus

et al. 1997

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A stable-isotope assay was used to analyze the effectiveness of various perdeuterated short-chain acyl coenzyme A (acyl-CoA) compounds as starter units for straight-and branched-chain fatty acid biosynthesis in cell extracts of Streptomyces collinus. In these extracts perdeuterated isobutyryl-CoA was converted to isopalmitate (a branched-chain fatty acid), while butyryl-CoA was converted to palmitate (a straight-chain fatty acid). These observations are consistent with previous in vivo analyses of fatty acid biosynthesis in S. collinus, which suggested that butyryl-CoA and isobutyryl-CoA function as starter units for palmitate and isopalmitate biosynthesis, respectively. Additionally, in vitro analysis demonstrated that acetyl-CoA can function as a starter unit for palmitate biosynthesis. Palmitate biosynthesis and isopalmitate biosynthesis in these cell extracts were both effectively inhibited by thiolactomycin, a known type II fatty acid synthase inhibitor. In vivo experiments demonstrated that concentrations of thiolactomycin ranging from 0.1 to 0.2 mg/ml produced both a dramatic decrease in the cellular levels of branched-chain fatty acids and a surprising three-to fivefold increase in the cellular levels of the straight-chain fatty acids palmitate and myristate. Additional in vivo incorporation studies with perdeuterated butyrate suggested that, in accord with the in vitro studies, the biosynthesis of the palmitate from butyryl-CoA decreases in the presence of thiolactomycin. In contrast, in vivo incorporation studies with perdeuterated acetate demonstrated that the biosynthesis of palmitate from acetylCoA increases in the presence of thiolactomycin. These observations clearly demonstrate that isobutyryl-CoA is a starter unit for isopalmitate biosynthesis and that either acetyl-CoA or butyryl-CoA can be a starter unit for palmitate biosynthesis in S. collinus. However, the pathway for palmitate biosynthesis from acetyl-CoA is less sensitive to thiolactomycin, and it is suggested that the basis for this difference is in the initiation step.

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Section: Discussionmentioning

confidence: 77%

Section: Discussionmentioning

confidence: 99%

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In vivo and in vitro effects of thiolactomycin on fatty acid biosynthesis in Streptomyces collinus

et al. 1997

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“…It appears that fabH can be deleted without causing lethality only when a second fabH copy is expressed in the same cells, implying that fabH is involved in an essential primary metabolic process, most likely fatty acid biosynthesis. This result does not rule out alternative mechanisms for initiation of fatty acid biosynthesis in S. coelicolor (e.g., any of the homologues of FabH that have been identified as part of the S. coelicolor genome project; a separate acetyl-CoA:ACP acyltransferase might bypass the action of FabH, as is the case in plant FASs [7]; decarboxylation of malonyl-ACP might provide an acetyl starter unit for straight-chain fatty acid biosynthesis, and a second FAS might also exist [5]). It merely shows that, if they exist, their activities are insufficient to suppress the effect of a deletion of fabH.…”

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confidence: 99%

β-Ketoacyl Acyl Carrier Protein Synthase III (FabH) Is Essential for Fatty Acid Biosynthesis in Streptomyces coelicolor A3(2)

et al. 2001

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The Streptomyces coelicolor fab (fatty acid biosynthesis) gene cluster (fabD-fabH-acpP-fabF) is cotranscribed to produce a leaderless mRNA transcript. One of these genes, fabH, encodes a ketoacyl synthase III that is essential to and is proposed to be responsible for initiation of fatty acid biosynthesis in S. coelicolor.Streptomyces spp. synthesize the majority of their fatty acids from branched starters such as isobutyryl, isovaleryl, and anteisovaleryl units to give odd-and even-numbered fatty acids with a methyl branch at the -terminus (80 to 90% of total fatty acid content); the remainder are synthesized from straight starters such as acetyl and butyryl units (11,21). The fatty acid synthase (FAS) of Streptomyces spp. is, like that found in many other bacteria (including the best-studied example, that of Escherichia coli), a type II or dissociable system (13, 18). The type II FAS consists of several discrete proteins that form loose associations to synthesize the fatty acid. The assembly of fatty acids is initiated by the condensation of an acyl coenzyme A (acyl-CoA) starter unit and a malonyl-acyl carrier protein (malonyl-ACP) extender unit; this condensation is catalyzed by ␤-ketoacyl ACP synthase III (FabH), the product of the fabH gene. In vitro biochemical studies suggest that FabH determines the choice of starter unit to be used. E. coli FabH is specific for an acetyl-CoA starter unit, whereas Bacillus subtilis and Streptomyces glaucescens FabHs can accept a broader range of substrates, including branched-and straight-chain units (2,8). In the case of the S. glaucescens FabH, the order of reactivity towards the different starters is isobutyryl-CoA Ͼ butyryl-CoA Ͼ acetyl-CoA. If FabH were solely responsible for the initiation of fatty acid biosynthesis in Streptomyces species, then one could hypothesize that its biochemical activity, together with the relative pool sizes of the different starter units in vivo, would account for the mix of branched and straight fatty acids (8). But when S. glaucescens was grown in the presence of high concentrations (480 M) of the FAS inhibitor thiolactomycin, branched-chain fatty acid biosynthesis was inhibited and the proportion of straight-chain fatty acids increased (50% inhibitory concentration [IC 50 ] for purified S. glaucescens FabH 20 M) (8). This result may be interpreted as evidence for a second; FabH-independent mechanism for fatty acid initiation in Streptomyces spp. Based on this second hypothesis, fabH should be dispensable to Streptomyces spp. In this study we provide further biochemical evidence for the role of a small cluster of presumed fab genes (which includes fabH) in Streptomyces coelicolor and a transcriptional analysis of the fab cluster, and we have attempted to disrupt fabH to determine if it is essential for the viability of the cells.The acpP gene product stimulates long-chain fatty acid biosynthesis in vitro. The S. coelicolor FAS is still relatively poorly understood; a cluster of four fab-like genes has been identified on the S. coelicol...

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“…The biosynthesis of erythromycin and other polyketides shows important similarities (25) to the more familiar process of fatty acid biosynthesis, and this has led to renewed interest in the structure, organization, and mechanism of action of fatty acid synthase in S. erythraea and in Streptomyces spp. Initial reports indicated that S. erythraea (39) and Streptomyces coelicolor (17) might contain a type I fatty acid synthase complex like that of Mycobacterium smegmatis (9), Brevibacterium ammoniagenes (30), or Saccharomyces cerevisiae (43,48), in which multifun.ctional polypeptides are tightly associated in a complex of high molecular weight. More recently, a small discrete acyl carrier protein (ACP) has been identified and purified from S. erythraea (21) on the basis of its ability to stimulate the incorporation of malonylCoA into acyl-ACP in a cell-free system.…”

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Cloning, characterization, and high-level expression in Escherichia coli of the Saccharopolyspora erythraea gene encoding an acyl carrier protein potentially involved in fatty acid biosynthesis

Revill

Leadlay

1991

J Bacteriol

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The erythromycin A-producing polyketide synthase from the gram-positive bacterium Saccharopolyspora erythraea (formerly Streptomyces erythraeus) has evident structural similarity to fatty acid synthases, particularly to the multifunctional fatty acid synthases found in eukaryotic cells. Fatty acid synthesis in S. erythraea has previously been proposed to involve a discrete acyl carrier protein (ACP), as in most prokaryotic fatty acid synthases. We have cloned and sequenced the structural gene for this ACP and find that it does encode a discrete small protein. The gene lies immediately adjacent to an open reading frame whose gene product shows sequence homology to known ,I-ketoacyl-ACP synthases. A convenient expression system for the S. erythraea ACP was obtained by placing the gene in the expression vector pT7-7 in Escherichia coli. In this system the ACP was efficiently expressed at levels 10 to 20% of total cell protein. The recombinant ACP was active in promoting the synthesis of branched-chain acyl-ACP species by extracts of S. erythraea. Electrospray mass spectrometry is shown to be an excellent method for monitoring the efficiency of in vivo posttranslational modification of ACPs.The macrolide antibiotic erythromycin A is produced by the gram-positive filamentous bacterium Saccharopolyspora erythraea (formerly Streptomyces erythraeus [32]), starting from simple fatty acyl-coenzyme A (CoA) precursors. The biosynthesis of erythromycin and other polyketides shows important similarities (25) to the more familiar process of fatty acid biosynthesis, and this has led to renewed interest in the structure, organization, and mechanism of action of fatty acid synthase in S. erythraea and in Streptomyces spp. Initial reports indicated that S. erythraea (39) and Streptomyces coelicolor (17) might contain a type I fatty acid synthase complex like that of Mycobacterium smegmatis (9), Brevibacterium ammoniagenes (30), or Saccharomyces cerevisiae (43, 48), in which multifun.ctional polypeptides are tightly associated in a complex of high molecular weight. More recently, a small discrete acyl carrier protein (ACP) has been identified and purified from S. erythraea (21) on the basis of its ability to stimulate the incorporation of malonylCoA into acyl-ACP in a cell-free system. This implies that the fatty acid synthase of S. erythraea may be patterned on the type II system found generally in prokaryotes, consisting of freely dissociable, monofunctional enzyme components. The best-studied system of this kind is that of Escherichia coli (see reference 52 for a recent review), but a discrete ACP is also apparently involved in fatty acid synthase from gram-positive bacteria more closely related to S. erythraea (1, 27) and which, like S. erythraea, characteristically produce iso-and anteiso-terminally branched fatty acids. Alternatively, there may exist more than one type of fatty acid synthase activity, as in Euglena gracilis (13). The recent (11) finding that the erythromycin-producing polyketide synthase of S. erythraea is a ...

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Partial purification of fatty acid synthetase from Streptomyces coelicolor

Cited by 13 publications

References 0 publications

In vivo and in vitro effects of thiolactomycin on fatty acid biosynthesis in Streptomyces collinus

In vivo and in vitro effects of thiolactomycin on fatty acid biosynthesis in Streptomyces collinus

β-Ketoacyl Acyl Carrier Protein Synthase III (FabH) Is Essential for Fatty Acid Biosynthesis in Streptomyces coelicolor A3(2)

Cloning, characterization, and high-level expression in Escherichia coli of the Saccharopolyspora erythraea gene encoding an acyl carrier protein potentially involved in fatty acid biosynthesis

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