Overproduction, purification and characterization of FgaPT2, a dimethylallyltryptophan synthase from Aspergillus fumigatus

• Published in: Microbiology (Society for General Microbiology) Vol. 151; no. 5; pp. 1499 - 1505
• Main Authors: Unsold, Inge A, Li, Shu-Ming
• Format: Journal Article
• Language: English
• Published: Reading Soc General Microbiol 01.05.2005; Society for General Microbiology
• Subjects: Alkyl and Aryl Transferases - chemistry; Alkyl and Aryl Transferases - genetics; Alkyl and Aryl Transferases - isolation & purification; Alkyl and Aryl Transferases - metabolism; Aspergillus fumigatus; Aspergillus fumigatus - enzymology; Aspergillus fumigatus - genetics; Biological and medical sciences; Cloning, Molecular; Drug Industry - methods; Ergot Alkaloids - biosynthesis; Ergot Alkaloids - chemistry; Fundamental and applied biological sciences. Psychology; Growth, nutrition, metabolism, transports, enzymes. Molecular biology; Magnetic Resonance Spectroscopy; Microbiology; Molecular Sequence Data; Mycology; Saccharomyces cerevisiae; Saccharomyces cerevisiae - enzymology; Saccharomyces cerevisiae - genetics; Sequence Analysis, DNA; Tryptophan - metabolism; Fungi; Alkaloid; Purification; Enzyme; Transferases; Dimethylallyltranstransferase; HPLC chromatography; Tryptophan; Fungi Imperfecti; Synthase; Aspergillus fumigatus; Thallophyta
• DOI: 10.1099/mic.0.27759-0
• ISSN: 1350-0872

Pharmazeutische Biologie, Pharmazeutisches Institut, Eberhard-Karls-Universität Tübingen, Auf der Morgenstelle 8, 72076 Tübingen, Germany Correspondence Shu-Ming Li shuming.li{at}uni-tuebingen.de A putative dimethylallyltryptophan synthase gene, fgaPT2 , was identified in the genome sequence of Aspergillus fumigatus . fgaPT2 was cloned and overexpressed in Saccharomyces cerevisiae . The protein FgaPT2 was purified to near homogeneity and characterized biochemically. This enzyme was found to convert L -tryptophan to 4-dimethylallyltryptophan, a reaction known to be the first step in ergot alkaloid biosynthesis. FgaPT2 is a soluble, dimeric protein with a subunit size of 52 kDa, and contains no putative prenyl diphosphate binding site (N/D)DXXD. K m values for L -tryptophan and dimethylallyl diphosphate (DMAPP) were determined as 8 and 4 µM, respectively. Metal ions, such as Mg 2+ and Ca 2+ , enhance the reaction velocity, but are not essential for the enzymic reaction. FgaPT2 showed a relatively strict substrate specificity for both tryptophan and DMAPP. FgaPT2 is the first enzyme in the biosynthesis of ergot alkaloids to be purified and characterized in homogeneous form after heterologous overproduction. Abbreviations: DMAPP, dimethylallyl diphosphate; DMAT, 4-dimethylallyltryptophan; DMATS, dimethylallyltryptophan synthase; FPP, farnesyl diphosphate; 6-methyl-DMAT, 4-dimethylallyl-6-methyltryptophan