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

Abstract

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-