Flavonol biosynthesis by nonheme iron dioxygenases: A computational study into the structure and mechanism.

Abstract

Plants produce flavonol compounds for vital functions regarding plant growth, fruit and flower colouring as well as fruit ripening processes. Several of these biosynthesis steps are stereo- and regioselective and are being carried out by nonheme iron enzymes. Using density functional theory calculations on a large active site model complex of flavanone-3β-hydroxylase (FHT), we established the mechanism for conversion of naringenin to its dihydroflavonol, which is a key step in the mechanism of flavonol biosynthesis. The reaction starts with dioxygen binding to the iron(II) centre and a reaction with α-ketoglutarate co-substrate gives succinate, an iron(IV)-oxo species and CO2 with large exothermicity and small reaction barriers. The rate-determining reaction step in the mechanism; however, is hydrogen atom abstraction of an aliphatic CH bond by the iron(IV)-oxo species. We identify a large kinetic isotope effect for the replacement of the transferring hydrogen atom by deuterium. In a final step the OH and substrate radicals combine to form the alcohol product with a barrier of several kcal mol-1. We show that the latter is the result of geometric constraints in the active site pocket. Furthermore, the calculations show that a weak tertiary CH bond is shielded from the iron(IV)-oxo species in the substrate binding position and therefore the enzyme is able to activate a stronger CH bond. As such, the flavanone-3β-hydroxylase enzyme reacts regioselectively with one specific CH bond of naringenin by avoiding activation of weaker bonds through tight substrate and oxidant positioning.