title: Site-directed mutagenesis switching a dimethylallyl tryptophan synthase to a specific tyrosine C3-prenylating enzyme.
abstract: The tryptophan prenyltransferases FgaPT2 and 7-DMATS (7-dimethylallyl tryptophan synthase) from Aspergillus fumigatus catalyze C(4)- and C(7)-prenylation of the indole ring, respectively. 7-DMATS was found to accept l-tyrosine as substrate as well and converted it to an O-prenylated derivative. An acceptance of l-tyrosine by FgaPT2 was also observed in this study. Interestingly, isolation and structure elucidation revealed the identification of a C(3)-prenylated l-tyrosine as enzyme product. Molecular modeling and site-directed mutagenesis led to creation of a mutant FgaPT2_K174F, which showed much higher specificity toward l-tyrosine than l-tryptophan. Its catalytic efficiency toward l-tyrosine was found to be 4.9-fold in comparison with that of non-mutated FgaPT2, whereas the activity toward l-tryptophan was less than 0.4% of that of the wild-type. To the best of our knowledge, this is the first report on an enzymatic C-prenylation of l-tyrosine as free amino acid and altering the substrate preference of a prenyltransferase by mutagenesis.
authors.affiliation: From the Institut für Pharmazeutische Biologie und Biotechnologie, Philipps-Universität Marburg, 35037 Marburg and.
authors.forename: Aili
authors.initials: A
authors.lastname: Fan
authors.affiliation: the Interfakultäres Institut für Biochemie, Eberhard Karls Universität Tübingen, 72076 Tübingen, Germany.
authors.forename: Georg
authors.initials: G
authors.lastname: Zocher
authors.affiliation: From the Institut für Pharmazeutische Biologie und Biotechnologie, Philipps-Universität Marburg, 35037 Marburg and.
authors.forename: Edyta
authors.initials: E
authors.lastname: Stec
authors.affiliation: the Interfakultäres Institut für Biochemie, Eberhard Karls Universität Tübingen, 72076 Tübingen, Germany.
authors.forename: Thilo
authors.initials: T
authors.lastname: Stehle
authors.affiliation: From the Institut für Pharmazeutische Biologie und Biotechnologie, Philipps-Universität Marburg, 35037 Marburg and shuming.li@staff.uni-marburg.de.
authors.forename: Shu-Ming
authors.initials: SM
authors.lastname: Li
mesh_terms: D019883:Alkyl and Aryl Transferases / Q000737:chemistry*; D001232:Aspergillus fumigatus / Q000201:enzymology*; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D005656:Fungal Proteins / Q000737:chemistry; D045782:Hemiterpenes / Q000737:chemistry; D007211:Indoles / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009943:Organophosphorus Compounds / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D054876:Prenylation; D015202:Protein Engineering / Q000379:methods; D014364:Tryptophan / Q000737:chemistry; D014367:Tryptophan Synthase / Q000737:chemistry*; D014443:Tyrosine / Q000737:chemistry*
chemical_list: D005656:Fungal Proteins; D045782:Hemiterpenes; D007211:Indoles; D009943:Organophosphorus Compounds; C043060:3,3-dimethylallyl pyrophosphate; D014443:Tyrosine; D014364:Tryptophan; D019883:Alkyl and Aryl Transferases; C032409:tryptophan dimethylallyltransferase; D014367:Tryptophan Synthase
keywords: Alkylation; Aspergillus; Dimethylallyl Tryptophan Synthase; Enzyme Catalysis; Enzyme Mutation; In Vitro Mutagenesis; Natural Product Biosynthesis; Protein Engineering; Tyrosine C3-Prenyltransferase