Reengineering a Tryptophan Halogenase To Preferentially Chlorinate a Direct Alkaloid Precursor

Installing halogens onto natural products can generate compounds with novel or improved properties. Notably, enzymatic halogenation is now possible as a result of the discovery of several classes of halogenases; however, applications are limited because of the narrow substrate specificity of these enzymes. Here we demonstrate that the flavin-dependent halogenase RebH can be engineered to install chlorine preferentially onto tryptamine rather than the native substrate tryptophan. Tryptamine is a direct precursor to many alkaloid natural products, including approximately 3000 monoterpene indole alkaloids. To validate the function of this engineered enzyme in vivo, we transformed the tryptamine-specific RebH mutant (Y455W)into the alkaloid-producing plant Madagascar periwinkle (Catharanthus roseus)and observed the de novo production of the halogenated alkaloid 12-chloro-19, 20-dihydroakuammicine. While wild-type (WT)RebH has been integrated into periwinkle metabolism reviously, the resulting tissue cultures accumulated substantial levels of 7-chlorotryptophan. Tryptophan decarboxylase, the enzyme that converts tryptophan to tryptamine, accepts 7-chlorotryptophan at only 3