| Structural highlights
Function
NOTF_ASPSM Deoxybrevianamide E synthase; part of the gene cluster that mediates the biosynthesis of notoamide, a fungal indole alkaloid that belongs to a family of natural products containing a characteristic bicyclo[2.2.2]diazaoctane core (PubMed:20722388). The first step of notoamide biosynthesis involves coupling of L-proline and L-tryptophan by the bimodular NRPS notE, to produce cyclo-L-tryptophan-L-proline called brevianamide F (PubMed:20722388). The reverse prenyltransferase notF then acts as a deoxybrevianamide E synthase and converts brevianamide F to deoxybrevianamide E via reverse prenylation at C-2 of the indole ring leading to the bicyclo[2.2.2]diazaoctane core (PubMed:20722388). Deoxybrevianamide E is further hydroxylated at C-6 of the indole ring, likely catalyzed by the cytochrome P450 monooxygenase notG, to yield 6-hydroxy-deoxybrevianamide E (Probable). 6-hydroxy-deoxybrevianamide E is a specific substrate of the prenyltransferase notC for normal prenylation at C-7 to produce 6-hydroxy-7-prenyl-deoxybrevianamide, also called notoamide S (PubMed:20722388). As the proposed pivotal branching point in notoamide biosynthesis, notoamide S can be diverted to notoamide E through an oxidative pyran ring closure putatively catalyzed by either notH cytochrome P450 monooxygenase or the notD FAD-linked oxidoreductase (Probable). This step would be followed by an indole 2,3-epoxidation-initiated pinacol-like rearrangement catalyzed by the notB FAD-dependent monooxygenase leading to the formation of notoamide C and notoamide D (PubMed:22188465). On the other hand notoamide S is converted to notoamide T by notH (or notD), a bifunctional oxidase that also functions as the intramolecular Diels-Alderase responsible for generation of (+)-notoamide T (Probable). To generate antipodal (-)-notoaminide T, notH' (or notD') in Aspergillus versicolor is expected to catalyze a Diels-Alder reaction leading to the opposite stereochemistry (Probable). The remaining oxidoreductase notD (or notH) likely catalyzes the oxidative pyran ring formation to yield (+)-stephacidin A (Probable). The FAD-dependent monooxygenase notI is highly similar to notB and is predicted to catalyze a similar conversion from (+)-stephacidin A to (-)-notoamide B via the 2,3-epoxidation of (+)-stephacidin A followed by a pinacol-type rearrangement (Probable). Finally, it remains unclear which enzyme could be responsible for the final hydroxylation steps leading to notoamide A and sclerotiamide (Probable).[1] [2] [3]
Publication Abstract from PubMed
Prenyltransfer is an early-stage carbon-hydrogen bond (C-H) functionalization prevalent in the biosynthesis of a diverse array of biologically active bacterial, fungal, plant, and metazoan diketopiperazine (DKP) alkaloids. Toward the development of a unified strategy for biocatalytic construction of prenylated DKP indole alkaloids, we sought to identify and characterize a substrate-permissive C2 reverse prenyltransferase (PT). As the first tailoring event within the biosynthesis of cytotoxic notoamide metabolites, PT NotF catalyzes C2 reverse prenyltransfer of brevianamide F. Solving a crystal structure of NotF (in complex with native substrate and prenyl donor mimic dimethylallyl S-thiolodiphosphate (DMSPP)) revealed a large, solvent-exposed active site, intimating NotF may possess a significantly broad substrate scope. To assess the substrate selectivity of NotF, we synthesized a panel of 30 sterically and electronically differentiated tryptophanyl DKPs, the majority of which were selectively prenylated by NotF in synthetically useful conversions (2 to >99%). Quantitative representation of this substrate library and development of a descriptive statistical model provided insight into the molecular origins of NotF's substrate promiscuity. This approach enabled the identification of key substrate descriptors (electrophilicity, size, and flexibility) that govern the rate of NotF-catalyzed prenyltransfer, and the development of an "induced fit docking (IFD)-guided" engineering strategy for improved turnover of our largest substrates. We further demonstrated the utility of NotF in tandem with oxidative cyclization using flavin monooxygenase, BvnB. This one-pot, in vitro biocatalytic cascade enabled the first chemoenzymatic synthesis of the marine fungal natural product, (-)-eurotiumin A, in three steps and 60% overall yield.
Data Science-Driven Analysis of Substrate-Permissive Diketopiperazine Reverse Prenyltransferase NotF: Applications in Protein Engineering and Cascade Biocatalytic Synthesis of (-)-Eurotiumin A.,Kelly SP, Shende VV, Flynn AR, Dan Q, Ye Y, Smith JL, Tsukamoto S, Sigman MS, Sherman DH J Am Chem Soc. 2022 Oct 26;144(42):19326-19336. doi: 10.1021/jacs.2c06631. Epub , 2022 Oct 12. PMID:36223664[4]
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.
References
- ↑ Ding Y, de Wet JR, Cavalcoli J, Li S, Greshock TJ, Miller KA, Finefield JM, Sunderhaus JD, McAfoos TJ, Tsukamoto S, Williams RM, Sherman DH. Genome-based characterization of two prenylation steps in the assembly of the stephacidin and notoamide anticancer agents in a marine-derived Aspergillus sp. J Am Chem Soc. 2010 Sep 15;132(36):12733-40. PMID:20722388 doi:10.1021/ja1049302
- ↑ Li S, Finefield JM, Sunderhaus JD, McAfoos TJ, Williams RM, Sherman DH. Biochemical characterization of NotB as an FAD-dependent oxidase in the biosynthesis of notoamide indole alkaloids. J Am Chem Soc. 2012 Jan 18;134(2):788-91. PMID:22188465 doi:10.1021/ja2093212
- ↑ Li S, Anand K, Tran H, Yu F, Finefield JM, Sunderhaus JD, McAfoos TJ, Tsukamoto S, Williams RM, Sherman DH. Comparative analysis of the biosynthetic systems for fungal bicyclo[2.2.2]diazaoctane indole alkaloids: the (+)/(-)-notoamide, paraherquamide and malbrancheamide pathways. Medchemcomm. 2012 Aug;3(8):987-996. PMID:23213353 doi:10.1039/C2MD20029E
- ↑ Kelly SP, Shende VV, Flynn AR, Dan Q, Ye Y, Smith JL, Tsukamoto S, Sigman MS, Sherman DH. Data Science-Driven Analysis of Substrate-Permissive Diketopiperazine Reverse Prenyltransferase NotF: Applications in Protein Engineering and Cascade Biocatalytic Synthesis of (-)-Eurotiumin A. J Am Chem Soc. 2022 Oct 26;144(42):19326-19336. PMID:36223664 doi:10.1021/jacs.2c06631
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