Site directed mutagenesis of Catharanthus roseus (+)-vincadifformine 19-hydroxylase (CYP71BY3) results in two distinct enzymatic functions. (September 2022)

• Record Type: Journal Article
• Authors: Williams, Danielle; Brzezinski, Weronika; Gordon, Heather; De Luca, Vincenzo
• Is Part Of: Phytochemistry. Volume 201(2022)
• Publication Date: 2022-09
• Subjects: Catharanthus roseus -- Apocynaceae -- Madagascar periwinkle -- Monoterpene indole alkaloids -- Cytochrome P450 -- (+)-vincadifformine 19-hydroxylase (V19H) -- tabersonine 19-hydroxylase (T19H) -- tabersonine 3-oxygenase (T3O) -- Homology-template modelling -- Site-directed mutagenesis; Botanical chemistry -- Periodicals; Biochemistry -- Periodicals; Botany -- Periodicals; Chimie végétale -- Périodiques; 572.2
• DOI: 10.1016/j.phytochem.2022.113265
• Languages: English
• ISSNs: 0031-9422

Abstract

Abstract: The most abundant monoterpenoid indole alkaloids (MIAs) in Catharanthus roseus roots include lochnericine and (+)-echitovenine. The formation of (+)-echitovenine involves a 3-step pathway including (+)-vincadifformine-19-hydroxylase (V19H) that differentiates it from a parallel pathway involved in the formation of lochnericine, hörhammericine and its O -acetylated derivative. Homology based modeling and docking experiments in the present study show that (+) and (−) vincadifformine can occupy the V19H active site and is proven experimentally by showing that (−)-vincadifformine is a competitive inhibitor of V19H. Comparative modeling of V19H with tabersonine 3-oxidase (T3O) and tabersonine 19-hydroxylase (T19H) that accept (−)-aspidosperma MIAs identified four conserved amino acid residues in T3O and T19H that were different in the V19H binding site and were used to generate a series of single-, double-, or four-point mutations in V19H. While all mutants retained their ability to convert (+)-vincadifformine to (+)-minovincinine only the four-point mutant gained T3O activity enabling it to convert (−)-tabersonine to tabersonine 2, 3-epoxide. The gain of T3O-like activity following mutagenesis without the loss of V19H activity supports the hypothesis that V19H shares a common ancestor to T3O which is involved in vindoline biosynthesis in C. roseus leaves. Graphical abstract: Replacement of 4-amino acids (L106R–S313T-A376P–F377L) in (+)-vindadifformine-19-hydroxylase (V19H) in results in acquisition of tabersonine 3-oxidase activity. Image 1 Highlights: (−)-vincadifformine is a reversible competitive inhibitor of (+)-vincadifformine 19-hydroxylase. Homology modelling of CYP71BY3 identify amino acids that alter MIA substrate specificity. Site-directed mutagenesis of CYP71BY3 results in tabersonine 3-oxygenase (T3O) activity. Evolution of V19H and T3O involves selection at least 4 amino acids from a common ancestor.