Reengineering a tryptophan halogenase to preferentially chlorinate a direct alkaloid precursor

Glenn, Weslee S., Nims, Ezekiel and O'Connor, Sarah E. (2011) Reengineering a tryptophan halogenase to preferentially chlorinate a direct alkaloid precursor. Journal of the American Chemical Society, 133 (48). pp. 19346-19349. ISSN 0002-7863

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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.

Item Type:	Article
Faculty \ School:	Faculty of Science > School of Biological Sciences Faculty of Science > School of Chemistry
UEA Research Groups:	Faculty of Science > Research Groups > Synthetic Chemistry (former - to 2017)
Depositing User:	Rhiannon Harvey
Date Deposited:	21 Mar 2012 15:27
Last Modified:	09 Mar 2024 22:30
URI:	https://ueaeprints.uea.ac.uk/id/eprint/38417
DOI:	10.1021/ja2089348

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