Overproduction, purification and characterization of FgaPT2, a dimethylallyltryptophan synthase from Aspergillus fumigatus.
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. Km values for L-tryptophan and dimethylallyl diphosphate (DMAPP) were determined as 8 and 4 microM, respectively. Metal ions, such as Mg2+ and Ca2+, 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.
Unsöld
Inge A
IA
Pharmazeutische Biologie, Pharmazeutisches Institut, Eberhard-Karls-Universität Tübingen, Auf der Morgenstelle 8, 72076 Tübingen, Germany.
Li
Shu-Ming
SM
Pharmazeutische Biologie, Pharmazeutisches Institut, Eberhard-Karls-Universität Tübingen, Auf der Morgenstelle 8, 72076 Tübingen, Germany.
D019883:Alkyl and Aryl Transferases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D001232:Aspergillus fumigatus / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D004345:Drug Industry / Q000379:methods; D004876:Ergot Alkaloids / Q000096:biosynthesis / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D017422:Sequence Analysis, DNA; D014364:Tryptophan / Q000378:metabolism
D004876:Ergot Alkaloids; D014364:Tryptophan; D019883:Alkyl and Aryl Transferases; C032409:tryptophan dimethylallyltransferase