(439d) A Novel Fungal Halogenase for Natural Products Biosynthesis

More than 4,500 halogenated natural products have been discovered from nature, many of which have been found to possess significant biological activities, such as chloramphenicol (antibacterial), vancomycin (antibacterial), and rebeccamycin (anticancer). Flavin-dependent halogenases represent a major group of enzymes that are involved in biological halogenations in natural products biosynthesis. However, only few halogenases have been identified so far, mostly prokaryotic tryptophan halogenases and substrate specific, which has hampered our understanding of this type of enzymes and their utilization for making novel bioactive molecules.

A fungal halogenase Rdc2 involved in radicicol biosynthesis in Pochonia chlamydosporia has been characterized and reconstituted in Escherichia coli. In presence of a partner flavin reductase Fre from E. coli, Rdc2 catalyzes chlorination of monocillin IV at C-13, confirming that Rdc2 is the dedicated flavin-dependent halogenase in the radicicol biosynthetic pathway. The enzyme has displayed broad substrate specificity towards various macrolactones such as dihydroresorcylide, and a structurally distinct plant natural product curcumin. In addition, Rdc2 was found to be a dihalogenase that can further halogenate the remaining aromatic proton in 11-chlorodihydroresorcylide to yield the corresponding dichlorinated derivative. Further investigation into its specificity towards halogen donors revealed that this enzyme is also a brominase that can catalyze mono- and dibromination of dihydroresorcylide to give rise to new brominated products. Kinetics studies were conducted to understand the two chlorination steps. Rdc2 represents a potent biocatalyst for preparation of halogenated derivatives from various bioactive natural products.