Algal neurotoxin biosynthesis repurposes the terpene cyclase structural fold into an N -prenyltransferase

Abstract

Prenylation is a common biological reaction in all domains of life wherein prenyl diphosphate donors transfer prenyl groups onto small molecules as well as large proteins. The enzymes that catalyze these reactions are structurally distinct from ubiquitous terpene cyclases that, instead, assemble terpenes via intramolecular rearrangements of a single substrate. Herein, we report the structure and molecular details of a new family of prenyltransferases from marine algae that repurposes the terpene cyclase structural fold for theN-prenylation of glutamic acid during the biosynthesis of the potent neurochemicals domoic acid and kainic acid. We solved the X-ray crystal structure of the prenyltransferase found in domoic acid biosynthesis, DabA, and show distinct active site binding modifications that remodel the canonical magnesium (Mg2+)-binding motif found in terpene cyclases. We then applied our structural knowledge of DabA and a homologous enzyme from the kainic acid biosynthetic pathway, KabA, to reengineer their isoprene donor specificities (geranyl diphosphate [GPP] versus dimethylallyl diphosphate [DMAPP]) with a single amino acid change. While diatom DabA and seaweed KabA enzymes share a common evolutionary lineage, they are distinct from all other terpene cyclases, suggesting a very distant ancestor to the larger terpene synthase family.

Toxicity of the Diatom Genus Pseudo-nitzschia (Bacillariophyceae): Insights from Toxicity Tests and Genetic Screening in the Northern Adriatic Sea

“…In any case, it appears that the ancestral state had the ability to produce DA, which also explains the occurrence of this toxin and its analogs in some other genera of diatoms and even red algae [42][43][44]. This is unless the biosynthetic apparatus was acquired during evolutionary history by horizontal gene transfer mechanisms perhaps on multiple occasions, as was suggested by [45] and which is the presumed pathway for the acquisition of saxitoxin production in dinoflagellates [46]. Horizontal gene transfer in protists is perhaps a neglected phenomenon and may be exacerbated by widespread viral infections [47].…”

Recent trends in biocatalysis

“…This N -PT belongs to the new family of PTs from marine algae of which the crystal structure and the catalytic mechanism have been revealed very recently. 478 The KGDOX can directly convert prekainic acid to kainic acid in the presence of α-ketoglutarate (αKG), l -ascorbate and Fe 3+ with 46% yield by purified enzyme and 57% yield using a recombinant E. coli . This enzymatic conversion solved the synthetic challenge of the stereocontrolled formation of the trisubstituted pyrrolidine ring and can replace the current chemical synthesis route with many steps.…”

Alternative metabolic pathways and strategies to high-titre terpenoid production inEscherichia coli

“…214 A distant relationship between prenyltransferases and terpenoid cyclases was identied recently that revealed a new family of prenyltransferases, which uses a repurposed terpenoid cyclase structural fold to prenylate glutamic acid. 215 Other prenyltransferases generated different diphosphate molecules, including chlorinated analogues, molecules having a hydrophilic moiety in their alkyl chain, or cyclic diphosphate molecules in previous in vitro studies, [216][217][218][219] and were reviewed recently. 220 Because these irregular and modied prenyl diphosphates differ structurally from their native counterparts, they could be used to create orthogonal pathways to terpenoids in E. coli, or expand the end-product range.…”