Detlef Gröger

MOLECULAR CLONING AND HETEROLOGOUS EXPRESSION OF ACRIDONE SYNTHASE FROM ELICITED RUTA GRAVEOLENS L. CELL SUSPENSION CULTURES

Cell suspension cultures of Ruta graveolens L. produce a variety of acridone alkaloids, and the accumulation can be stimulated by the addition of fungal elicitors. Acridone synthase, the enzyme catalyzing the synthesis of 1,3-dihydroxy-N-methylacridone from N-methylanthraniloyl-CoA and malonyl-CoA, had been isolated from these cells, and the partial enzyme polypeptide sequence, elucidated from six tryptic fragments, revealed homology to heterologous chalcone synthases. Poly(A)+ RNA was isolated from Ruta cells that had been treated for 6 h with a crude cell wall elicitor from Phytophthora megasperma f. sp. glycinea, and a cDNA library was constructed in λ2AP. Clones harboring acridone synthase cDNA were isolated from the library by screening with a synthetic oligonucleotide probe complementary to a short stretch of sequence of the enzyme peptide with negligible homology to chalcone synthases. The identity of the clones was substantiated by DNA sequencing and by recognition of five additional peptides, determined previously from tryptic acridone synthase digests, in the translated sequence. An insert of roughly 1.4 kb encoded the complete acridone synthase, and alignments at both DNA and protein levels corroborated the high degree of homology to chalcone synthases. Expression of the enzyme in vector pET-11c in the Escherichia coli pLysS host strain proved the identity of the cloned cDNA. The heterologous enzyme in the crude E. coli extract exhibited high acridone but no chalcone synthase activity. The results were fully supported by northern blot hybridizations which revealed that the specific transcript abundance did not increase but rather decreased upon white light irradiation of cultured Ruta graveolens L. cells, a condition that commonly induces the abundance of chalcone synthase transcripts.

Biosynthesis of indoxyl derivatives in Isatis tinctoria and Polygonum tinctorium

Abstract L -[5- 3 H]Tryptophan was administered to young plants of Isatis tinctoria and Polygonum tinctorium . Labelled indoxyl derivatives could be isolated from stems and roots of both plant species. Stems of I. tinctoria are able to convert [ 3 H]-labelled tryptophan into indican and isatan B. Root cultures of I. tinctoria synthesize indican and isatan B and incorporate biosynthetic precursors into indoxyl derivatives.

Differential regulation and distribution of acridone synthase in Ruta graveolens

Cell suspension cultures of Ruta graveolens L. accumulate polyketide metabolites such as acridone alkaloids and flavonoid pigments. Whereas flavonoid synthesis is induced by light, the production of alkaloids can be enhanced in dark-cultured cells by treatment with fungal elicitors. Acridone synthase (ACS) catalyzes the committed condensing reaction of acridone biosynthesis yielding 1,3-dihydroxy-N-methylacridone from N-methylanthraniloyl- and malonyl-CoAs. The reaction proceeds in a manner analogous to that of chalcone synthase (CHS) which catalyzes the first committed step in flavonoid biosynthesis and cDNA and protein sequences of Ruta ACS possess a high degree of sequence homology to heterologous CHSs. ACS transcript abundance and specific activity were monitored in cultured R. graveolens cells irradiated either continuously with white light or treated with fungal elicitor over a period of 24 h and found to increase transiently upon elicitor treatment and to decrease upon light irradiation. Immunodetection with a rabbit polyclonal ACS antiserum revealed that the amounts of ACS polypeptide decreased slightly in light-irradiated cells but increased in elicitor-treated Ruta cells. Fluorescence microscopy and tissue print hybridizations were employed to aid in localizing the sites of storage and biosynthesis of acridone alkaloids in Ruta plants. Yellow fluorescing alkaloids were detected particularly in root tissue adjacent to the rhizodermis, but also in the endodermis and vascular tissue of the hypocotyl. ACS transcript abundance in situ followed the same spatial pattern, indicating that the synthesis of acridones likely proceeds at all sites of deposition rather than exclusively in the root. Expression in planta and the induction response of ACS suggest that the alkaloids serve as phytoanticipins or phytoalexins in the defense of Ruta particularly to soil-borne pathogens or as feeding deterrents.

Zur Biosynthese von Ergotoxinalkaloiden in Claviceps purpurea

Summary The biosynthetic pathway of ergotoxine alkaloids (ergocornine, ergokryptine) was studied with Claviceps purpurea strain Pepty 695. Lysergic acid-U-14C is specifically incorporated into the lysergic acid moiety of ergotoxins. Lysergic acid amide is no intermediate in the formation of peptide type ergot alkaloids. The specific incorporation of leucine-U-14C into ergocornine is much lower than in ergokryptine. Leucine-U-14C labels preferentially the leucine part of ergokryptine but to an appreciable amount also the α-hydroxyvaline portion of this particular alkaloid. The main part of radioactivity after leucine-14C administration in ergocornine is located in the valine moiety. A series of experiments using valine-14C and valine-14C,15N were performed in order to clarify the role of this amino acid in the formation of the peptide portion of ergotoxins. Using differently labeled valine preparations and varying the feeding-time we observed that the α-hydroxyvaline part of ergocornine had always an higher specific radioactivity than the valine moiety. Thus earlier results were confirmed and lends support to the view that the formation of the peptide chain starts from the proline end. In ergokryptine about 2/3 of the radioactivity was located in the α-hydroxy-amino acid portion and about 25 % in the leucine part. After administration of valine-14C,15N the 15N/14C ratio in theiX-hydroxyvaline part was measured. In different experiments the 15N/14C ratio varied considerably. This seems to indicate an high L-valinetransaminase activity. Nevertheless we assume that the nitrogen of the α-hydroxy amino acid moiety in ergotoxine alkaloids is derived from valine.

Synthesis of 1,3-dihydroxy-N-methylacridone and its conversion to rutacridone by cell-free extracts of Ruta graveolens cell cultures

Abstract Acridone synthase was isolated from cell suspension cultures of Ruta graveolens which catalysed the formation of 1,3-dihydroxy-N- methylacridone from N-methylanthraniloyl-CoA and malonyl-CoA. No cofactors were required for this enzyme reaction. Potassium phosphate buffer was superior compared to Tris-HCl. Sodium ascorbate instead of mercaptoethanol as oxidation protectant showed an advantageous effect on acridone synthase activity. The enzyme was strongly inhibited by 1,3-dihydroxy-N-methylacridone and by the antibiotic cerulenin. Microsomal preparations from Ruta graveolens cell suspension cultures catalysed an NADPH- and oxygen-dependent condensation of 1,3-dihydroxy-N- methylacridone and isopentenyl pyrophosphate. The reaction product was identified as rutacridone. Mg2+ or Mn2+ ions were necessary for optimal rutacridone synthase activity. The enzyme was inhibited by a number of inhibitors of cytochrome P-450 enzymes. A prenylated acridone, viz. glycocitrine-II was identified as an essential intermediate. Under in vivo conditions glycocitrine-II is incorporated into rutacridone, but a clear-cut conversion of glycocitrine-II by microsomal preparations (cyclase) was not observed. Microsomes converted rutacridone into furofoline-I. A number of detergents was used for solubilization of membrane-bound proteins of Ruta microsomes. Highest specific glycocitrine -II synthase (prenyltransferase) activity was obtained after solubilization with dodecylmaltoside.

Purification and properties of acridone synthase from cell suspension cultures of Ruta graveolens L.

Acridone synthase has been purified from cell suspension cultures of Ruta graveolens using a combination of gel filtration and ion exchange chromatography. The purified enzyme has an apparent molecular weight of 69 kDa on gel filtration and a subunit structure on SDS-PAGE of 40 kDa. The apparent Km-values are 10.64 μM and 32.8 μM for N-methylanthraniloyl-CoA and malonyl-CoA, respectively. Tryptic digestion of the homogeneous acridone synthase was performed. Seven of the peptides were chosen for microsequencing. The homology of the amino acid sequences from this particular polypeptide and corresponding peptides from chalcone synthase 3 from garden pea amounted to 76%.

Biosynthesis of acridone alkaloids formation of rutacridone by cell-free extracts of Ruta graveolens cell suspension cultures

Microsomes prepared either by ultracentrifugation or MgCl2 precipitation from cultured Ruta graveolens cells catalyze the condensation of 1,3‐dihydroxy‐N‐methylacridone and isopentenylpyrophosphate or dimethylallylpyrophosphate. In the presence of NADPH and oxygen rutacridone was identified as reaction product. By omission of NADPH glycocitrine‐II is accumulated. The results suggest that at first a prenylated acridone is formed which in turn is cyclized giving the dihydrofuran part of rutacridone.

Synthese und Stoffwechsel von Lysergylpeptiden in einem Ergotoxine bildenden Claviceps-Stamm

Summary Radioactive d-lysergyl-L-valyl-L-leucine-OMe, d-lysergyl-L-valyl-L-valine-OMe and d-lysergyl-valyl-valyl-proline-OMe were synthesized according to the dicyclohexylcarbodiimide/1-hydroxybenzotriazole procedure. The d-lysergyl- and d-isolysergyl peptide esters were separated by DC1). The lysergyl peptides and peptide derivatives were characterized by melting point, optical rotation, UV, mass spectrometry, and chromatographic methods. These peptides are split into their components after feeding to the mycelium of Claviceps purpurea, strain Pepty 695. Also crude enzyme preparations of the same strain catalyze the degradation of the lysergyl peptides. Radioactivity of d-lysergyl-Val-Val-Pro-OMe is incorporated by intact mycelium into ergotoxine alkaloids only after breakdown of the precursor. Apparently it does not serve as free intermediate in ergocornine biosynthesis. It might be concluded that the whole process of cyclol alkaloid formation in ergot takes place on a multienzyme complex.

Acetohydroxyacid synthase from cell suspension cultures of Isatis tinctoria L. and Ruta graveolens L.

Summary Acetohydroxyacid synthase (EC 4.1.3.18) has been extracted from cell suspension cultures of Isatis tinctoria (Cruciferae) and Ruta graveolens (Rutaceae). A combination of salt precipitation, gel filtration and ion exchange chromatography was used for partial purification. The apparent molecular masses of AHAS were Mr 82,000 and 85,000 for Isatis and Ruta, respectively. FAD was an absolute requirement for AHAS activity. The apparent Km values of Isatis-AHAS are the following ones: FAD 6,3 × 10−6 M; TPP 6,3 × 10−6 M; pyruvate 7 × 10−3, and 6 × 10−3 M (for Ruta-AHAS). Branched-chain amino acids and chlorsulfuron are feedback inhibitors for Isatis-AHAS but acetohydroxyacid synthase from Ruta is not sensitive to valine, leucine and isoleucine.