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. 2020 Sep 1;21(17):2449-2454.
doi: 10.1002/cbic.202000004. Epub 2020 May 14.

Flavin-Dependent Monooxygenases NotI and NotI' Mediate Spiro-Oxindole Formation in Biosynthesis of the Notoamides

Amy E Fraley  1   2 , Hong T Tran  1   3 , Samantha P Kelly  1   3 , Sean A Newmister  1 , Ashootosh Tripathi  1   2 , Hikaru Kato  4 , Sachiko Tsukamoto  4 , Lei Du  5 , Shengying Li  5 , Robert M Williams  6 , David H Sherman  1   2   7   8
Affiliations

Flavin-Dependent Monooxygenases NotI and NotI' Mediate Spiro-Oxindole Formation in Biosynthesis of the Notoamides

Amy E Fraley et al. Chembiochem. .

Abstract

The fungal indole alkaloids are a unique class of complex molecules that have a characteristic bicyclo[2.2.2]diazaoctane ring and frequently contain a spiro-oxindole moiety. While various strains produce these compounds, an intriguing case involves the formation of individual antipodes by two unique species of fungi in the generation of the potent anticancer agents (+)- and (-)-notoamide A. NotI and NotI' have been characterized as flavin-dependent monooxygenases that catalyze epoxidation and semi-pinacol rearrangement to form the spiro-oxindole center within these molecules. This work elucidates a key step in the biosynthesis of the notoamides and provides an evolutionary hypothesis regarding a common ancestor for production of enantiopure notoamides.

Keywords: alkaloids; biosynthesis; flavin monooxygenase; notoamide; spirocycle.

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Conflict of interest statement

Conflict of Interest

The authors declare no conflict of interest.

Figures

Figure 1.
Figure 1.
Biochemical activity assays with NotI and NotI′. (a) (+)-versicolamide B (8) standard. (b) (+)- and (−)-notoamide B (5 and 10) standards. (c) NotI′ reaction with (−)-6-epi-stephacidin A (7). (d) NotI reaction with 7. (e) No enzyme control with 7. (f) NotI′ reaction with (+)-6-epi-stephacidin A (6). (g) NotI reaction with 6. (h) No enzyme control with 6. (i) NotI′ reaction with (−)-stephacidin A (4). (j) NotI reaction with 4. (k) No enzyme control with 4. (l) NotI′ reaction with (+)-stephacidin A (9). (m) NotI reaction with 9. (n) No enzyme control with 9. No enzyme controls are shown in purple, reactions with NotI are shown in blue, and NotI′ reactions are shown in red. The HPLC data were collected at a wavelength of 240 nm.
Figure 2.
Figure 2.
Alignment of PhqK (PDB ID: 6pvi) and Phyre2 models of NotI and NotI′ demonstrating the presence of the catalytic arginine in all three proteins. FAD conformation was modelled from urate oxidase HpxO (PDB ID: 3rp7).
Scheme 1.
Scheme 1.
Reactions catalyzed by NotI/NotI′ in vitro and in the respective native fungal species.
Scheme 2.
Scheme 2.
Proposed (−)/(+)-notoamide A (19 and 20) biosynthetic pathways. The functions of enzymes highlighted in red have been experimentally confirmed. The terminal N-hydroxylation to generate 19 and 20 is catalyzed by an additional enzyme subsequent to spirocyclization.
Scheme 3.
Scheme 3.
Proposed mechanism for epoxidation and spirocyclization catalysed by NotI and NotI′.
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