Enzymatic biosynthesis of ergotamine and investigation on some aminoacyl-tRNA synthetases in Claviceps

Maier, W.; Krauspe, R.; Gröger, D.

doi:10.1016/0168-1656(85)90016-1

Cited by 4 publications

(7 citation statements)

References 23 publications

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“…It may be noted that work on the total cell-free synthesis of ergopeptines (Maier et al, 1981(Maier et al, , 1983(Maier et al, , 1985 yielded results that are quite different from those presented here. The most striking difference is that these authors found ergopeptine synthesis dependent on the biosynthetic precursors of D-lysergic acid, namely, agroclavine or elymoclavine, rather than on D-lysergic acid itself.…”

Section: Analysis Of Radioactive Lysergyl Peptides Besidescontrasting

confidence: 99%

D-Lysergic acid activation and cell-free synthesis of D-lysergyl peptides in an enzyme fraction from the ergot fungus Claviceps purpurea

Keller¹,

Han²,

Stöffler‐Meilicke³

1988

Biochemistry

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The D-lysergic acid activating enzyme from the ergot fungus Claviceps purpurea was purified to near homogeneity. It has a native Mr of about 245,000 and in its denatured form is a single polypeptide chain of Mr 62,000. The enzyme catalyzes the ATP-pyrophosphate exchange reaction dependent on D-lysergic acid and, though much less, that dependent on dihydrolysergic acid. Western blot analysis of SDS electropherograms of crude protein extracts from C. purpurea using monospecific antibodies directed against the D-lysergic acid activating enzyme revealed the immunostaining of one particular band which was identical with that of the D-lysergic acid activating enzyme. No significant immunoreactive band with higher molecular weight was seen, which precludes the possibility that the enzyme had arisen from the proteolysis of a high molecular weight ergot peptide synthetase. An ammonium sulfate fractionated enzyme fraction was prepared from C. purpurea strain C1 that catalyzed the incorporation of D-lysergic acid into two peptides which besides D-lysergic acid contained alanine, phenylalanine, and proline. Dihydrolysergic acid was efficiently incorporated into the corresponding dihydrolysergic acid containing analogues of the two compounds. Radiochemical analysis and degradation studies suggest that the two D-lysergic acid containing peptides most probably are N-[N-(D-lysergyl)-L-alanlyl]-L-phenylalanyl-L-proline lactam and N-[N-(D-lysergyl)-L-analyl]-L-phenylalanyl-D-proline lactam, respectively. N-[N-(D-Lysergyl)-L-alanyl]-L-phenylalanyl-L-proline lactam is considered to be the immediate precursor of ergotamine.(ABSTRACT TRUNCATED AT 250 WORDS)

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Section: Analysis Of Radioactive Lysergyl Peptides Besidescontrasting

confidence: 99%

D-Lysergic acid activation and cell-free synthesis of D-lysergyl peptides in an enzyme fraction from the ergot fungus Claviceps purpurea

Keller¹,

Han²,

Stöffler‐Meilicke³

1988

Biochemistry

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“…In addition, the question as to which chemical form of the precursor of peptide-bound D-lysergic acidsi.e., either free D-lysergic acid, D-lysergyl Coenzyme A, or a simple clavinesmay serve as substrate in the assembly process remained a central controversial issue in these investigations. [152][153][154] Eventually the development of a cell-free system that incorporated exclusively free D-lysergic acid into the novel noncyclol peptide D-lysergyl-L-alanyl-L-phenylalanyl-L-proline lactam (Figure 14) provided the starting point to study D-lysergic acid peptide formation at the molecular level. 155 D-Lysergyl-L-alanyl-L-phenylalanyl-L-proline lactam has long been considered the immediate precursor of ergotamine.…”

Section: Ergot Peptide Alkaloidsmentioning

confidence: 99%

“…Various models for the mechanism of assembly of ergopeptines, elaborated from cell-free and in vivo data, have been proposed in the past. [152][153][154] They suggest unusual direction of peptide chain growth or free precursor peptides in the assembly of the Dlysergyl peptide backbonessuch as free D-lysergylalanine or D-lysergylvaline reacting with preformed prolyldiketopiperazinessobscuring the reaction mechanism of alkaloid cylopeptide formation. [152][153][154]162 Analysis of peptide intermediates accumulating on LPS 1 and LPS 2 in the presence of D-lysergic acid (or dihydrolysergic acid) and the amino acids of the alkaloid peptide chains shed light on the mode of the assembly of D-lysergyl peptides.…”

Section: Mechanism Of Formation Of D-lysergyl Peptide Lactammentioning

confidence: 99%

“…[152][153][154] They suggest unusual direction of peptide chain growth or free precursor peptides in the assembly of the Dlysergyl peptide backbonessuch as free D-lysergylalanine or D-lysergylvaline reacting with preformed prolyldiketopiperazinessobscuring the reaction mechanism of alkaloid cylopeptide formation. [152][153][154]162 Analysis of peptide intermediates accumulating on LPS 1 and LPS 2 in the presence of D-lysergic acid (or dihydrolysergic acid) and the amino acids of the alkaloid peptide chains shed light on the mode of the assembly of D-lysergyl peptides. 163 D-lysergic acid activated by LPS 2 was strictly required for priming peptide formation on LPS 1, to which it is transferred in the presence of the amino acid which occupies position I of the alkaloid cyclopeptide, e.g., alanine in Figure 14.…”

Section: Mechanism Of Formation Of D-lysergyl Peptide Lactammentioning

confidence: 99%

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Multifunctional Peptide Synthetases

H¹,

Keller²,

Vater³

et al. 1997

Chem. Rev.

234

147

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“…There was no ratio of 1 : 1 between covalently bound Phe to non-covalently bound phenylalanine as in the case of peptide antibiotics formation (LIPMANN 1971). This may be easily explained by the fact that ergotamine synthetase and phenylalanyl-tRNA synthetase are not separable by the used ion exchange chromatography (MAIER et al 1985). Thus most of the TCA-labile phenylalanine is attached to the Phe-tRNA synthetase.…”

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confidence: 99%

Formation of enzyme‐bound L‐phenylalanine by a cell‐free system of Claviceps purpurea

Maier

Gröger

1985

J. Basic Microbiol.

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Ergotamine synthetase purified by ammonium sulfate precipitation, gel filtration and ion exchange chromatography on DEAE-Sepharose CL-6B binds L-phenylalanine in an acid-stable, apparently covalent, form. This substrate-enzyme complex might be involved in ergopeptine synthesis.Peptide ergot alkaloids (ergopeptines) are unique natural products bearing a cyclol structure. These modified tripeptides result from the reaction of an a-hydroxy-aamino acid adjacent to lysergic acid with the carboxyl carbon of proline (FLOSS 1976 al. 1981, 1983). If ergopeptines are synthesized by a nucleic acid free mechanism involving a peptide-forming multienzyme, covalently-bound intermediates, e.g. activated amino acids may occur. We have tested this possibility using labelled phenylalanine.Mycelium of ergot strains J a p 471/I (ergotamine forming) grown for 4 days in a sucrose/mannitol/ ammonium citrate medium was used for enzyme preparation (MAIER et al. 1985). This was done by grinding lyophilized cells with dry ice in a mortar in the presence of a 0.2 M Tris-HCI buffer p H 7.8 containing 40% glycerol; 20 mM KCl; 10 mM MgCl . 6 H,O; 10 mix dithioerythritol, 2 mM phenylmethylsulfonyl fluoride and 1 mM EDTA (buffer A). The suspension were centrifuged a t 20000 x g for 30 min. The supernatant was fractionatedby slow addition of a (NH,),SO, solution to 67% saturation. The precipitate was collected by centrifugation, dissolved in 0.1 M Tris-HC1 buffer (pH 7.8) and layered onto a Sepharose 4B column (3.5 x 50 em). The elution was performed with a glycerol containing 0.1 M Tris-HCi (pH 7.8) buffer. To the pooled ergotamine synthetase containing fractions ammonium sulfate was added to 67% saturation. The protein was collected by centrifugation, dissolved in 3 ml 0,05 M Tris-HC1 (pH 7.8) buffer and dialyzed for 14 h against 1 1 of the same buffer. The dialyzed enzyme was applied on a DEAE-Sepharose CL-6B column (2.4 x 20 cm) and eluted with 200 ml of a linear NaC1-gradient (0-0.5 M). The active fractions were pooled and the protein precipitated as described in the previous step.The assay for amino acid binding to the enzyme protein was performed as follows : The reaction mixture contained in a final volume of 0.5 ml: 400 pg protein, 2 pCi phenylalanine-~-[ring-2,6-SH] (48.3 Ci/mmol) or (1 Ci/mmol), respectively; 1 pmol ATP; 5 pmol MgC1, . 6 H,O and 300 p1 buffer A.The mixture was incubated 30 min a t 32 "C and after termination of the reaction 2 mg bovine serum albumin were added. The enzyme solution was placed on a Sephadex G-50 column (1 x 17 cm) equilibrated with 0.1 M Tris-HC1 (pH 7.8) buffer containing 10% glycerol, 2 mM 2-mercapto-

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Enzymatic biosynthesis of ergotamine and investigation on some aminoacyl-tRNA synthetases in Claviceps

Cited by 4 publications

References 23 publications

D-Lysergic acid activation and cell-free synthesis of D-lysergyl peptides in an enzyme fraction from the ergot fungus Claviceps purpurea

D-Lysergic acid activation and cell-free synthesis of D-lysergyl peptides in an enzyme fraction from the ergot fungus Claviceps purpurea

Multifunctional Peptide Synthetases

Formation of enzyme‐bound L‐phenylalanine by a cell‐free system of Claviceps purpurea

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