title: 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-(gamma,gamma-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 alpha 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 KM and Vmax were determined in the metal-free EDTA buffer (KMDMAPP, 14 microM; KML-tryptophan, 40 microM; Vmax, 215 nmol min-1 mg-1), 4 mM CaCl2 (KMDMAPP, 8.0 microM; KML-tryptophan, 17 microM; Vmax, 504 nmol min-1 mg-1), and 4 mM MgCl2 (KMDMAPP, 8.0 microM; KML-tryptophan, 12 microM; Vmax, 455 nmol min-1 mg-1). The product was isolated and characterized by 1H NMR, uv, and FAB mass spectrometry.
authors.affiliation: Department of Chemistry, University of Utah, Salt Lake City 84112.
authors.forename: J C
authors.initials: JC
authors.lastname: Gebler
authors.forename: C D
authors.initials: CD
authors.lastname: Poulter
mesh_terms: D019883:Alkyl and Aryl Transferases; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D002967:Claviceps / Q000201:enzymology* / Q000254:growth & development; D005612:Freeze Drying; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008970:Molecular Weight; D016339:Spectrometry, Mass, Fast Atom Bombardment; D013056:Spectrophotometry, Ultraviolet; D014166:Transferases / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D014166:Transferases; D019883:Alkyl and Aryl Transferases; C032409:tryptophan dimethylallyltransferase