title: Hydroxylation of the triterpenoid dipterocarpol with CYP106A2 from Bacillus megaterium.
abstract: The bacterial steroid-hydroxylase CYP106A2 from Bacillus megaterium ATCC 13368 hydroxylates a variety of 3-oxo-Δ-4-steroids and has recently been shown to catalyse regioselective hydroxylation of the diterpene abietic acid, as well as the pentacyclic triterpene 11-keto-β-boswellic acid. The broad substrate spectrum of this enzyme makes it an excellent candidate for biotechnological application. Because the natural substrate of this enzyme is not known, we assumed that the whole substrate spectrum might not yet be fully discovered. The difference spectroscopy method was used to screen a natural product library of 502 compounds. Screening of the library resulted in the identification of twelve hits, among them eight potential and four known substrates for CYP106A2. Interestingly, when testing the potential substrates, product formation was obtained only with triterpenes, namely dipterocarpol and betulin. Dipterocarpol is the most promising compound for biotechnological application because it is a dammarane-type triterpenoid, as are the major bioactive compounds of ginseng. The dipterocarpol hydroxylation products were analysed by NMR and identified as 7β-hydroxydipterocarpol and 7β,11α-dihydroxydipterocarpol. To investigate the putative bioactive properties of these novel compounds, in vitro cytotoxicity assays with HeLa and COS-1 cells were performed. The substrate dipterocarpol and the dihydroxylated product did not show cytotoxic activity in our study. By contrast, the 7β-hydroxylated product was found to be cytotoxic to both tested cell lines. This study highlights the potency of CYP106A2 as a versatile biocatalyst for the bioconversion of natural products into pharmaceutically relevant bioactive products.
authors.affiliation: Department of Biochemistry, Saarland University, Saarbruecken, Germany.
authors.forename: Daniela
authors.initials: D
authors.lastname: Schmitz
authors.forename: Josef
authors.initials: J
authors.lastname: Zapp
authors.forename: Rita
authors.initials: R
authors.lastname: Bernhardt
mesh_terms: D000818:Animals; D001410:Bacillus megaterium / Q000201:enzymology; D001426:Bacterial Proteins / Q000378:metabolism*; D019556:COS Cells; D002522:Chlorocebus aethiops; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D003603:Cytotoxins / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D006900:Hydroxylation; D014315:Triterpenes / Q000378:metabolism* / Q000633:toxicity
chemical_list: D001426:Bacterial Proteins; D003603:Cytotoxins; D014315:Triterpenes; D003577:Cytochrome P-450 Enzyme System; C517094:15beta-hydroxylase CYP106A2, Bacillus megaterium