title: Reengineering a tryptophan halogenase to preferentially chlorinate a direct alkaloid precursor.
abstract: 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 previously, 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% of the efficiency of the native substrate tryptophan, thereby creating a bottleneck. The RebH Y455W mutant circumvents this bottleneck by installing chlorine onto tryptamine, a downstream substrate. In comparison with cultures harboring RebH and WT RebF, tissue cultures containing mutant RebH Y455W and RebF also accumulate microgram per gram fresh-weight quantities of 12-chloro-19,20-dihydroakuammicine but, in contrast, do not accumulate 7-chlorotryptophan, demonstrating the selectivity and potential utility of this mutant in metabolic engineering applications.
authors: Department of Biological Chemistry, The John Innes Centre, Norwich NR4 7UH, UK.
authors: Weslee S
authors: WS
authors: Glenn
authors: Ezekiel
authors: E
authors: Nims
authors: Sarah E
authors: SE
authors: O'Connor
mesh_terms: D000470:Alkaloids / Q000378:metabolism*; D030008:Catharanthus / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D054879:Halogenation; D006576:Heterocyclic Compounds, 4 or More Rings / Q000378:metabolism; D060847:Metabolic Engineering; D009154:Mutation; D010088:Oxidoreductases / Q000235:genetics* / Q000378:metabolism*; D015202:Protein Engineering; D014363:Tryptamines / Q000378:metabolism*; D014364:Tryptophan / Q000031:analogs & derivatives / Q000378:metabolism*
chemical_list: C412590:19,20-dihydroakuammicine; D000470:Alkaloids; D006576:Heterocyclic Compounds, 4 or More Rings; D014363:Tryptamines; C037586:7-chlorotryptophan; C030820:tryptamine; D014364:Tryptophan; D010088:Oxidoreductases; C407363:tryptophan halogenase