Biosynthesis of anthracyclines: carminomycin 4-O-methyltransferase, the terminal enzymic step in the formation of daunomycin

Connors, Neal; Bartel, Paul L.; Strohl, William R.

doi:10.1099/00221287-136-9-1895

Cited by 26 publications

(32 citation statements)

References 15 publications

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“…We previously found that a methyltransferase activity in crude cell extracts of several daunomycin-producing streptomycetes methylated carminomycin and 13-dihydrocarminomycin (Fig. I), but no other glycones or aglycones tested, suggesting that conversion of carminomycin to daunomycin is the terminal reaction in daunomycin biosynthesis (Connors et al, 1990b). Here we report the partial purification and characteristics of an enzyme from Streptomyces sp.…”

Section: Introductionmentioning

confidence: 99%

“…strain C5 and other daunomycin-producing streptomycetes, an enzyme capable of methylating carminomycin to a product that co-migrated with daunomycin (Connors et al, 1990b). In those studies, AdoMet of very high specific activity [46 Ci (mol 14C-AdoMet)-'] was used to detect the activity in crude cell extracts (Connors et al, 1990b).…”

Section: Analysis Of Cmt Activitymentioning

confidence: 99%

“…the formation of 8-rhodomycinone from the theoretical polyketide precursor, has now been characterized in relative detail (Eckardt & Wagner, 1988;Bartel et al, 1990;Connors et al, 1990a;Strohl & Connors, 1992). On the other hand, except for our characterization of TDP-D-glucose 4,6-dehydratase, the first enzyme in TDP-daunosamine biosynthesis (Thompson et al, 1992), and our preliminary description of carminomycin methyltransferase (CMT) activity (Connors et al, 1990b), the enzymic reactions involved in glycosylation and the conversion of 8-rhodomycinone to daunomycin remain largely uncharacterized. We previously found that a methyltransferase activity in crude cell extracts of several daunomycin-producing streptomycetes methylated carminomycin and 13-dihydrocarminomycin (Fig.…”

Section: Introductionmentioning

confidence: 99%

“…strain C5 based on results obtained with blocked mutants (Bartel et al, 1990), in vitro bioconversion reactions (Connors et al, 1990a), and the discovery of an enzyme capable of methylating carminomycin (Connors et al, 1990b). The first half of the pathway, i.e.…”

Section: Introductionmentioning

confidence: 99%

“…Reactions carried out for the separation and determination of products by TLC and autoradiography, as well as for monitoring the early steps of CMT purification, contained S-aden~syl-[methyl-'~C]-~-methionine [14C-AdoMet ; 0.04 pCi (1.48 kBq) per reaction, 43 mCi mmol-' (1591 MBq mmol-'); New England Nuclear] instead of diluted 3H-AdoMet. Reaction mixtures to be analysed by TLC were solventextracted as described previously (Connors et al, 1990b), air dried and then applied in 10 p1 chloroform to a 0.25 mm layer of silica gel. The TLC plates (Whatman) were developed with chloroform/methanol/ glacial acetic acid/H,O (15 : 5 : 1 : 1, by vol.)…”

Section: Introductionmentioning

confidence: 99%

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Partial purification and properties of carminomycin 4-O-methyltransferase from Streptomyces sp. strain C5

Connors

Strohl²

1993

Journal of General Microbiology

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A methyltransferase that acts on carminomycin and 13-dihydrocarminornycin, and that is postulated to be the terminal enzyme in the daunomycin biosynthesis pathway, was purified to near-homogeneity from the daunomycinand baumycin-producing Streptomyces sp. strain C5. The enzyme was obtained in approximately 5 % yield with a purification of 114-fold in specific activity over the sample precipitated with 30-50 % ammonium sulphate. Polyacrylamide gel electrophoresis under denaturing conditions indicated a subunit M, of about 41 000. The enzyme was shown by gel filtration chromatography to have an M, of approximately 166000, suggesting that it is a homotetramer. Kinetic analysis indicated an affinity for S-adenosyl-L-methionine typical of antibiotic methyltransferases ; the enzyme had a slightly higher affinity for carminomycin than for 13-dihydrocarminomycin. The reaction product from methylation of carminomycin was confirmed by chromatography and mass spectral analysis to be daunomycin. The purified enzyme did not catalyse methylation of the aglycones carminomycinone or 13-dihydrocarminomycinone. S-Adenosyl-L-homocysteine inhibited the methyltransferase, whereas homocysteine, adenosine, adenine, &-rhodomycinone, daunomycin, and daunomycinone showed little or no inhibitory activity.

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Section: Introductionmentioning

confidence: 99%

Section: Analysis Of Cmt Activitymentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Partial purification and properties of carminomycin 4-O-methyltransferase from Streptomyces sp. strain C5

Connors

Strohl²

1993

Journal of General Microbiology

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Immobilization and properties of carminomycin 4‐O‐methyltranferase, the enzyme which catalyzes the final step in the biosynthesis of daunorubicin in Streptomyces peucetius

Scotti

1995

Biotech & Bioengineering

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The carminomycin 4-O-methyltransferase enzyme from Streptomyces peucetius was covalently immobilized on 3M Emphaze ABI-activated beads. Optimal conditions of time, temperature, pH, ionic strength, enzyme, substrate (carminomycin), and cosubstrate (S-adenosyl-L-methionine) concentrations were defined for the immobilization reaction. Protein immobilization yield ranged from 52% to 60%. Including carminomycin during immobilization had a positive effect on the activity of the immobilized enzyme but a strongly negative effect on the coupling efficiency. The immobilized enzyme retained at least 57% of its maximum activity after storage at 4 degrees C for more than 4 months. The properties of the free and immobilized enzyme were compared to determine whether immobilization could alter enzyme activity. Both soluble and bound enzyme exhibited the same pH profile with an optimum near 8.0. Immobilization caused an approximately 50% decrease in the apparent K(m) (K'(m)) for carminomycin while the K'(m) for S-adenosyl-L-methionine was approximately doubled. A 57% decrease in the V(max) value occurred upon immobilization. These changes are discussed in terms of active site modifications as a consequence of the enzyme immobilization. This system has a potential use in bioreactors for improving the conversion of carminomycin to daunorubicin. (c) 1995 John Wiley & Sons, Inc.

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Biosynthese der Anthracycline: eine Neuinterpretation der Ergebnisse zur Daunomycin‐Biosynthese

Wagner¹,

Eckardt²,

Ihn³

et al. 1991

J. Basic Microbiol.

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On the basis of literature data and our own experiments the "late" biosynthetic pathway to daunomycin has been interpreted from a new point of view considering both the in vivo biosynthesis and formation of shunt products. In contrast to existing hypotheses proposed by other authors we discuss a modified sequence leading to C-11 oxidation and, as a consequence, understand epsilon-rhodomycinone as a shunt product instead of a biosynthetic intermediate. In addition, a new hypothesis about the "early" steps of the ring formation from polyketides by a sequence of enzyme reactions has been proposed.

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Biosynthesis of anthracyclines: carminomycin 4-O-methyltransferase, the terminal enzymic step in the formation of daunomycin

Cited by 26 publications

References 15 publications

Partial purification and properties of carminomycin 4-O-methyltransferase from Streptomyces sp. strain C5

Partial purification and properties of carminomycin 4-O-methyltransferase from Streptomyces sp. strain C5

Immobilization and properties of carminomycin 4‐O‐methyltranferase, the enzyme which catalyzes the final step in the biosynthesis of daunorubicin in Streptomyces peucetius

Biosynthese der Anthracycline: eine Neuinterpretation der Ergebnisse zur Daunomycin‐Biosynthese

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