A small, discrete acyl carrier protein is involved in de novo fatty acid biosynthesis in <i>Streptomyces erythraeus</i>

Hale, Richard S.; Jordan, K N; Leadlay, Peter F.

doi:10.1016/0014-5793(87)80436-2

Cited by 28 publications

(20 citation statements)

References 17 publications

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“…Interestingly, only 16-and 14-carbon chain fatty acid products were detected in assays using a butyryl-CoA starter unit, regardless of the ratio of butyryl-CoA to malonyl-CoA. A previous analysis of fatty acid biosynthesis in Saccharopolyspora erythraea using an isobutyrylCoA starter unit showed that under the assay conditions the predominant product appeared to be a terminally branched eight-carbon fatty acid (8).…”

Section: Resultsmentioning

confidence: 84%

“…FAS assays have previously been employed for cell extracts of streptomycetes and other bacteria which produce predominantly branched-chain fatty acids. These methods typically utilize either a radioactive acyl-CoA starter unit or a malonyl-CoA extender and quantitate the radioactivity that extracts into an organic phase (8,16). Although sensitive, such assays do not permit the unambiguous determination of intact incorporation of starter units into the product.…”

Section: Resultsmentioning

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: Resultsmentioning

confidence: 84%

Section: Resultsmentioning

confidence: 99%

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

et al. 1997

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“…erythruea apparently bears a closer relationship to that of vertebrate fatty-acid synthases than to the S. erythraea fatty-acid synthase, which appears to be a type-I1 system of readily disaggregated proteins [42,431, patterned on the wellstudied fatty-acid synthase of E. coli (reviewed in [44]). The multifunctional 6-methylsalicylate synthase of P. patulum also bears far less resemblance in its sequence to the P. patulum fatty-acid synthase than it does to the vertebrate fatty-acid synthases, and this is reflected in its structural organisation [21].…”

Section: The Domain Organisation Of Debs 2 Andmentioning

confidence: 99%

6‐Deoxyerythronolide‐B synthase 2 from Saccharopolyspora erythraea

Bevitt

Cortés

Haydock

et al. 1992

European Journal of Biochemistry

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170

101

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Sequencing of the eryA region of the erythromycin biosynthetic gene cluster from Saccharopolyspora erythraea has revealed another structural gene (ORF B), in addition to the previously characterised ORF A , which appears to encode a component of 6-deoxyerythronolide-B synthase, the enzyme that catalyses the first stage in the biosynthesis of the polyketide antibiotic erythromycin A. The nucleotide sequence of ORF B, whch lies immediately adjacent to ORF A , has been determined. The predicted gene product of ORF B is a polypeptide of 374417 Da (3568 amino acids), which is highly similar to the product of ORF A and which likewise contains a number of separate domains, each with substantial amino acid sequence similarity to components of known fatty-acid synthases and polyketide synthases. The order of the predicted active sites along the chain from the N-terminus is 3-oxoacyl-synthase -acyltransferase -acyl-carrier-protein -3-oxoacyl-synthase -acyltransferase -dehydratase -enoylreductase -oxoreductase -acyl-carrier-protein. The position of the dehydratase active site has been pinpointed for the first time for any polyketide synthase or vertebrate fatty-acid synthase. The predicted domain structure of 6-deoxyerythronolide-B synthase is strikingly similar to that previously established for vertebrate fatty-acid synthases. This analysis of the sequence supports the view that the erythromycin-producing polyketide synthase contains three multienzyme polypeptides, each of which accomplishes two successive cycles of polyketide chain extension. In this scheme, the role of the ORF B gene product is to accomplish extension cycles 3 and 4.

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“…The overall degree of sequence identity between E. coli and S. erythraea fatty acid synthase ACPs, over 40%, is markedly higher than the degree of sequence identity (about 25%) between the potential S. erythraea fatty acid synthase ACP and any of the polyketide synthase ACPs (7,45) or ACP domains (11) that were tested. E. coli ACP can stimulate fatty acyl-ACP synthesis by cell extracts of S. erythraea that have been previously depleted of endogenous ACP (21), so the close similarity in primary structure evidently gives rise to a very similar three-dimensional structure. Expression of the S. erythraea ACP gene in E. coli gave an enzymatically active protein, indicating that the S. erythraea ACP is a substrate for the E. coli (holo)-ACP synthetase, which transfers the 4'-phosphopantetheine prosthetic group from CoA to the ACP.…”

Section: Pc T T V T T a C Gpv F G A T S A C Gvn Y S I S S A C G P I Kmentioning

confidence: 99%

“…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.…”

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confidence: 99%

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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A small, discrete acyl carrier protein is involved in de novo fatty acid biosynthesis in Streptomyces erythraeus

Cited by 28 publications

References 17 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

6‐Deoxyerythronolide‐B synthase 2 from Saccharopolyspora erythraea

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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