Reengineering a Tryptophan Halogenase To Preferentially Chlorinate a Direct Alkaloid Precursor
| Autor: | Sarah E. O'Connor, Ezelkiel Nims, Weslee S. Glenn |
|---|---|
| Rok vydání: | 2011 |
| Předmět: |
Tryptamine
Halogenation Catharanthus Stereochemistry Protein Engineering Heterocyclic Compounds 4 or More Rings Biochemistry Catalysis Metabolic engineering chemistry.chemical_compound Alkaloids Colloid and Surface Chemistry Biosynthesis heterocyclic compounds Indole test biology Tryptophan Substrate (chemistry) General Chemistry Catharanthus roseus biology.organism_classification Tryptamines Metabolic Engineering chemistry Mutation Oxidoreductases |
| Zdroj: | Journal of the American Chemical Society. 133:19346-19349 |
| ISSN: | 1520-5126 0002-7863 |
| DOI: | 10.1021/ja2089348 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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