Purification and characterization of dimethylallyl tryptophan synthase from Claviceps purpurea

Abstract

Dimethylallyl tryptophan synthase (DMAT synthase) catalyzes the alkylation of l-tryptophan by dimethylallyl diphosphate to form 4-(γ,γ-dimethylallyl)- l-tryptophan. The enzyme from mycelia of Claviceps purpurea was purified approximately 125-fold to apparent homogeneity by chromatography on n-butyl Sepharose, Q Sepharose, phenyl Sepharose, and Protein Pak as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Analysis by gel filtration chromatography and SDS-PAGE indicated that DMAT synthase is an α 2 dimer with a molecular mass of 105 kDa. The purified enzyme was active in metal-free buffer containing EDTA. However, activity was enhanced upon addition of divalent calcium or magnesium ions to the buffer. Values for K M and V max were determined in the metal-free EDTA buffer ( K M DMAPP, 14 μM; K M L-tryptophan, 40 μM; V max, 215 nmol min −1 mg −1), 4 m m CaCl 2 ( K M DMAPP, 8.0 μM; K M L-truptophan, 17 μM; V max, 504 nmol min −1 mg −1), and 4 m m MgCl 2 ( K M DMAPP, 8.0 μM; K M L-tryptophan, 12 μM; V max, 455 nmol min −1 mg −1). The product was isolated and characterized by 1H NMR, uv, and FAB mass spectrometry.