title: Bifunctional CYP81AA proteins catalyse identical hydroxylations but alternative regioselective phenol couplings in plant xanthone biosynthesis.
abstract: Xanthones are natural products present in plants and microorganisms. In plants, their biosynthesis starts with regioselective cyclization of 2,3',4,6-tetrahydroxybenzophenone to either 1,3,5- or 1,3,7-trihydroxyxanthones, catalysed by cytochrome P450 (CYP) enzymes. Here we isolate and express CYP81AA-coding sequences from Hypericum calycinum and H. perforatum in yeast. Microsomes catalyse two consecutive reactions, that is, 3'-hydroxylation of 2,4,6-trihydroxybenzophenone and C-O phenol coupling of the resulting 2,3',4,6-tetrahydroxybenzophenone. Relative to the inserted 3'-hydroxyl, the orthologues Hc/HpCYP81AA1 cyclize via the para position to form 1,3,7-trihydroxyxanthone, whereas the paralogue HpCYP81AA2 directs cyclization to the ortho position, yielding the isomeric 1,3,5-trihydroxyxanthone. Homology modelling and reciprocal mutagenesis reveal the impact of S375, L378 and A483 on controlling the regioselectivity of HpCYP81AA2, which is converted into HpCYP81AA1 by sextuple mutation. However, the reciprocal mutations in HpCYP81AA1 barely affect its regiospecificity. Product docking rationalizes the alternative C-O phenol coupling reactions. Our results help understand the machinery of bifunctional CYPs.
authors.affiliation: Institute of Pharmaceutical Biology, Technische Universität Braunschweig, Mendelssohnstraße 1, Braunschweig 38106, Germany.|Center of Pharmaceutical Engineering (PVZ), Technische Universität Braunschweig, Franz-Liszt-Straße 35A, Braunschweig 38106, Germany.
authors.forename: Islam
authors.initials: I
authors.lastname: El-Awaad
authors.affiliation: Institute of Biotechnology, RWTH Aachen University, Worringerweg 1, Aachen 52074, Germany.
authors.forename: Marco
authors.initials: M
authors.lastname: Bocola
authors.affiliation: Institute of Pharmaceutical Biology, Technische Universität Braunschweig, Mendelssohnstraße 1, Braunschweig 38106, Germany.|Center of Pharmaceutical Engineering (PVZ), Technische Universität Braunschweig, Franz-Liszt-Straße 35A, Braunschweig 38106, Germany.
authors.forename: Till
authors.initials: T
authors.lastname: Beuerle
authors.affiliation: Institute of Pharmaceutical Biology, Technische Universität Braunschweig, Mendelssohnstraße 1, Braunschweig 38106, Germany.|Center of Pharmaceutical Engineering (PVZ), Technische Universität Braunschweig, Franz-Liszt-Straße 35A, Braunschweig 38106, Germany.
authors.forename: Benye
authors.initials: B
authors.lastname: Liu
authors.affiliation: Institute of Pharmaceutical Biology, Technische Universität Braunschweig, Mendelssohnstraße 1, Braunschweig 38106, Germany.|Center of Pharmaceutical Engineering (PVZ), Technische Universität Braunschweig, Franz-Liszt-Straße 35A, Braunschweig 38106, Germany.
authors.forename: Ludger
authors.initials: L
authors.lastname: Beerhues
mesh_terms: D000595:Amino Acid Sequence; D055162:Biocatalysis; D053898:Biosynthetic Pathways / Q000235:genetics; D002478:Cells, Cultured; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006900:Hydroxylation; D020902:Hypericum / Q000166:cytology / Q000201:enzymology* / Q000235:genetics; D062105:Molecular Docking Simulation; D009154:Mutation; D019800:Phenol / Q000737:chemistry / Q000378:metabolism*; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013237:Stereoisomerism; D044004:Xanthones / Q000737:chemistry / Q000378:metabolism*
chemical_list: D010940:Plant Proteins; D044004:Xanthones; D019800:Phenol; D003577:Cytochrome P-450 Enzyme System