Further Studies on the Biosynthesis of Granaticin

He, Xian-Guo; Chang, Chandong; Chang, Ching Jer; Vederas, John C.; McInnes, A. G.; Walter, John A.; Floss, Heinz G.

doi:10.1515/znc-1986-1-232

Cited by 15 publications

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“…Such an observation is inconsistent with the use of either FabH or FadA to initiate TCM C biosynthesis directly from acetyl-CoA (Figure 2). A similar lack of intact incorporation of deuterium label from [2-13 C, 2 H 3 ]acetate has been reported for the starter unit of granaticin (22). This observation led to a suggestion of malonyl-CoA as a starter unit, which is decarboxylated by the "enzyme matrix".…”

Section: Resultssupporting

confidence: 66%

“…This observation led to a suggestion of malonyl-CoA as a starter unit, which is decarboxylated by the "enzyme matrix". In the granatacin study NMR analysis of the methyl group derived from the starter unit demonstrated no evidence of 13 C 2 H 3 but some level of 13 C 2 H 2 and 13 C 2 H species (22). In our study on TCM C we did not see any evidence for these species.…”

Section: Resultscontrasting

confidence: 65%

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Enzymes Involved in Fatty Acid and Polyketide Biosynthesis in Streptomyces glaucescens: Role of FabH and FabD and Their Acyl Carrier Protein Specificity

2002

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Malonyl acyl carrier protein (ACP) is used as an extender unit in each of the elongation steps catalyzed by the type II dissociated fatty acid synthase (FAS) and polyketide synthase (PKS) of Streptomyces glaucescens. Initiation of straight-chain fatty acid biosynthesis by the type II FAS involves a direct condensation of acetyl-CoA with this malonyl-ACP to generate a 3-ketobutyryl-ACP product and is catalyzed by FabH. In vitro experiments with a reconstituted type II PKS system in the absence of FabH have previously shown that the acetyl-ACP (generated by decarboxylation of malonyl-ACP), not acetyl-CoA, is used to initiate tetracenomycin C (TCM C) biosynthesis. We have shown that sgFabH activity is present in S. glaucescens fermentations during TCM C production, suggesting that it could contribute to initiation of TCM C biosynthesis in vivo. Isotope incorporation studies with [CD3]acetate and [13CD3]acetate demonstrated significant intact retention of three deuteriums into the starter unit of palmitate and complete washout of deuterium label into the starter unit of TCM C. These observations provide evidence that acetyl-CoA is not used directly as a starter unit for TCM C biosynthesis in vivo and argue against an involvement of FabH in this process. Consistent with this conclusion, assays of the purified recombinant sgFabH with acetyl-CoA demonstrated activity using malonyl-ACP generated from either FabC (the S. glaucescens FAS ACP) (k(cat) 42.2 min(-1), K(m) 4.5 +/- 0.3 microM) or AcpP (the E. coli FAS ACP) (k(cat) 7.5 min(-1), K(m) 6.3 +/- 0.3 microM) but not TcmM (the S. glaucescens PKS ACP). In contrast, the sgFabD which catalyzes conversion of malonyl-CoA to malonyl-ACP for fatty acid biosynthesis was shown to be active with TcmM (k(cat) 150 min(-1), K(m) 12.2 +/- 1.2 microM), AcpP (k(cat) 141 min(-1), K(m) 13.2 +/- 1.6 microM), and FabC (k(cat) 560 min(-1), K(m) 12.7 +/- 2.6 microM). This enzyme was shown to be present during TCM C production and could play a role in generating malonyl-ACP for both processes. Previous demonstrations that the purified PKS ACPs catalyze self-malonylation and that a FabD activity is not required for polyketide biosynthesis are shown to be an artifact of the expression and purification protocols. The relaxed ACP specificity of FabD and the lack of a clear alternative are consistent with a role of FabD in providing malonyl-ACP precursors for PKS as well as FAS processes. In contrast, the ACP specificity of FabH, isotope labeling studies, and a demonstrated alternative mechanism for initiation of the PKS process provide unequivocal evidence that FabH is involved only in the FAS process.

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Section: Resultssupporting

confidence: 66%

Section: Resultscontrasting

confidence: 65%

Enzymes Involved in Fatty Acid and Polyketide Biosynthesis in Streptomyces glaucescens: Role of FabH and FabD and Their Acyl Carrier Protein Specificity

2002

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“…Snipes et al 1979). Feeding experiments have established that granaticin is synthesized from eight molecules of acetate and one molecule of glucose (Snipes et al 1979;He et al 1986). 1).…”

Section: Introductionmentioning

confidence: 99%

Identification ofStreptomyces violaceoruber Tü22 genes involved in the biosynthesis of granaticin

et al. 1995

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A 50 kb region of DNA from Streptomyces violaceoruber Tü22, containing genes encoding proteins involved in the biosynthesis of granaticin, was isolated. The DNA sequence of a 7.3 kb fragment from this region, located approximately 10 kb from the genes that encode the polyketide synthetase responsible for formation of the benzoisochromane quinone skeleton, revealed five open reading frames (ORF1-ORF5). The deduced amino acid sequence of GraE, encoded by ORF2, shows 60.8% identity (75.2% similarity) to a dTDP-glucose dehydratase (StrE) from Streptomyces griseus. Cultures of Escherichia coli containing plasmids with ORF2, on a 2.1 kb BamHI fragment, were able to catalyze the formation of dTDP-4-keto-6-deoxy-D-glucose from dTDP-glucose at 5 times the rate of control cultures, confirming that ORF2 encodes a dTDP-glucose dehydratase. The amino acid sequence encoded by ORF3 (GraD) is 51.4% identical (69.9% similar) to that of StrD, a dTDP-glucose synthase from Streptomyces griseus. The amino acid sequence encoded by ORF4 shares similarities with proteins that confer resistance to tetracycline and methylenomycin, and is suggested to be involved in transporting granaticin out of the cells by an active efflux mechanism.

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“…nidulans group) (9). Another group of compounds comprises the benzoisochromane quinones, such as actinorhodin (10) and granaticin (1 l), whose relevant structural moieties (12) have been shown to be of polyketide origin by extensive biosynthetic studies (10,11). The latter compounds, however, are metabolites of actinomycetes, that is, prokaryotic organisms which may not be encompassed by the rule.…”

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Confirmation of the polyketide origin of kotanin by incorporation of [1,2-¹³C₂]acetate

Stothers¹,

Stoessl²

1988

Can. J. Chem.

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Incorporation experiments with [ l ,2-'3C2]acetate establish that the biosynthesis of kotanin, a dimeric 5-rnethylcoumarin, proceeds through a polyketide pathway and, therefore, represents an exception to Turner's rule regarding the relative lengths of the uncyclized residues of polyketide chains. The previous I3C signal assignments are also revised.J. B. STOTHERS ET ALBERT STOESSL. Can. J. Chern. 66 28 16 (1988).Des exp6riences d'incorporation du acetate [I3C2-1 ,2] ont permis de dtrnontrer que la biosynthtse de la kotanine, un dirntre de la mkthyl-5 cournarine, se produit par le biais d'un polycttide et qu'elle correspond donc i une exception h la rtgle de Turner concernant les longueurs relatives des rksidus non-cyclists des chaines polycttides. On a aussi revise les attributions antkrieures des signaux relatifs au 13c.[Traduit par la revue]Fungal metabolites whose structures are in real or apparent in the expectation of isolating orlandin (5) as the labelled breach of Turner's rule (1) that "the uncyclized residues from product. In a preliminary cold run, however, chromatography the methyl ends of polyketide chains are never shorter than of the culture extract furnished crystalline kotanin (3) in good residues from the carboxyl end of the chain" include marticin (1) yield. This compound had not been reported previously as a from Fusarium martii (2), colletruncoic acid methyl ester (2) metabolite of A. niger. Other fractions from the chromatogram from Colletotrichum truncatum (3), kotanin (3) and demethyl kotanin (4) from Aspergillus clavatus (4, 5), orlandin (5) from A. niger (6), siderin (6), the monomer of kotanin, from A. variecolor (7,8), and the desertorins A, B, and C (7-9, respectively) from Emericella desertorurn ( A . nidulans group) (9). Another group of compounds comprises the benzoisochromane quinones, such as actinorhodin (10) and granaticin (1 l), whose relevant structural moieties (12) have been shown to be of polyketide origin by extensive biosynthetic studies (10, 11). The latter compounds, however, are metabolites of actinomycetes, that is, prokaryotic organisms which may not be encompassed by the rule. Of the true fungal metabolites marticin has been proven (2, 12) to be of mixed polyketide and tricarboxylic acid cycle origin, which accounts for the apparent anomaly. Of the others, only siderin (6) has been the subject of an experimental biosynthetic study. It was found (7) that A. variecolor cultures incorporated sodium [2-14Cl]acetate into 6 with 0.75% efficiency. Kuhn-Roth degradation of the labelled siderin placed 25.8% of its total radioactivity into the 5-methyl group and the excess, over the expected result of uniform incorporation (20% at each of 5 sites), was very plausibly ascribed to the starter effect as frequently seen in polyketide biosynthesis. Unfortunately, lack of material precluded further degradative experiments that could have rendered this conclusion certain. Alternative interpretations of the data, therefore, could not be entirely discounted. For instance, it might b...

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Further Studies on the Biosynthesis of Granaticin

Cited by 15 publications

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Enzymes Involved in Fatty Acid and Polyketide Biosynthesis in Streptomyces glaucescens: Role of FabH and FabD and Their Acyl Carrier Protein Specificity

Enzymes Involved in Fatty Acid and Polyketide Biosynthesis in Streptomyces glaucescens: Role of FabH and FabD and Their Acyl Carrier Protein Specificity

Identification ofStreptomyces violaceoruber Tü22 genes involved in the biosynthesis of granaticin

Confirmation of the polyketide origin of kotanin by incorporation of [1,2-¹³C₂]acetate

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Further Studies on the Biosynthesis of Granaticin

Cited by 15 publications

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Enzymes Involved in Fatty Acid and Polyketide Biosynthesis in Streptomyces glaucescens: Role of FabH and FabD and Their Acyl Carrier Protein Specificity

Enzymes Involved in Fatty Acid and Polyketide Biosynthesis in Streptomyces glaucescens: Role of FabH and FabD and Their Acyl Carrier Protein Specificity

Identification ofStreptomyces violaceoruber Tü22 genes involved in the biosynthesis of granaticin

Confirmation of the polyketide origin of kotanin by incorporation of [1,2-13C2]acetate

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Confirmation of the polyketide origin of kotanin by incorporation of [1,2-¹³C₂]acetate