Biosynthetic allene and alkyne formation by enzymatic prenyl demethylation

Abstract Allenes and alkynes are versatile functional groups in chemical biology and drug discovery. However, how nature biosynthetically installs them in natural products, especially allenes, remains poorly understood. Here we uncovered that allenes and alkynes can be formed enzymatically through oxidative C( sp 2 )-demethylation of the common five-carbon prenyl group. Two fungal cytochrome P450 monooxygenases, PpnB and NseB, from the penipratynolene and sinuxylamide biosynthetic pathways, respectively, were shown to catalyze oxidative removal of a C( sp 2 )-methyl group in O -prenyl l -tyrosine to afford O -homoallenyl l -tyrosine and O -but-2-ynyl l -tyrosine, respectively. Combining density functional theory calculations and biochemical assays with isotopically labeled substrates, a mechanism involving selective C–C bond cleavage followed by product-determining hydrogen atom abstraction is presented. An additional P450 enzyme from the penipratynolene pathway, PpnD, acts as an oxidative isomerase that converts the four-carbon terminal allene into a terminal alkyne. This sophisticated enzymatic editing strategy to install allene and alkyne expands the catalytic repertoire of P450 enzymes.