Elucidating Cysteine-Assisted Synthesis of Indirubin by a Flavin-Containing Monooxygenase

Abstract

Indirubin is a biologically active compound found in Danggui Longhui Wan, which is a traditional Chinese medicine for chronic myelocytic leukemia. In the biosynthesis of indirubin, the formation of indigo, which is a stereoisomer of indirubin, is a major side reaction. Recent findings have suggested that cysteine supplementation shifts product selectivity from indigo to indirubin. Here, we disclose how cysteine is involved in enhancing the product selectivity in the synthesis of indirubin using a flavin-containing monooxygenase from Methylophaga aminisulfidivorans (MaFMO). First, cysteine reacts with indoxyl to synthesize 2-cysteinylindoleninone, inhibiting the dimerization of indoxyl. Second, the reducing power of cysteine allows MaFMO to additionally hydroxylate indoxyl toward isatin, overcoming the problem in biased distribution of two different precursors. Third, cysteine activates isatin to react with 2-cysteinylindoleninone to form indirubin. Based on this revealed mechanism, indirubin derivatives with different indole ring components were synthesized.

Constructing multi‐enzymatic cascade reactions for selective production of 6‐bromoindirubin from tryptophan in Escherichia coli

“…As an alternative to inhibit IG formation, cysteine (Cys) was used. Cys can provide reducing power as well as thiol moiety to retain 3‐OH‐indole and produce 2‐cysteinylindoleninone (2‐Cys‐indoleninone), which subsequently reacts with 6‐Br‐2‐oxindole to generate 6BrIR (J. Kim et al, 2019) (Supporting Information: Figure S12). Interestingly, when Cys was used in the reactions, 6BrIR was produced without IG irrespective of the ratio of 6‐Br‐2‐oxindole to 3‐OH‐indole (Figure 5a).…”

“…The in situ generated 3‐OH‐indole and 2‐Cys‐indoleninone react with 6‐Br‐2‐oxindole to produce 6BrIR. However, the remaining 3‐OH‐indole is oxygenated again by MaFMO to become isatin, and the produced isatin reacts with 3‐OH indole leading to IR byproduct (J. Kim et al, 2019). Therefore, the whole‐cell reaction conditions of ΔtnaA TnaA+MaFMO to generate 6BrIR without forming IR were further examined.…”

“…pH variations were performed by adding 2 M Tris‐HCl buffer to make the desired pH, and the pH of the reactions were checked before starting the reactions. According to the previous report, the purification and in vitro reaction conditions of MaFMO were performed (J. Kim et al, 2019).…”

“…In the case of indole oxidation, several oxygenases have been reported for the production of indigoids, since IG and IR are produced from the dimerization of C3‐C3′‐oxygenated indoles and C3‐C2′‐oxygenated indoles, respectively (Choi, 2020; Fabara & Fraaije, 2020). However, most oxygenases including MaFMO (Loncar et al, 2019), CYP102G4 (J. Kim et al, 2018), and naphthalene dioxygenase (Cheng et al, 2016) preferentially oxygenate the C3 position of the indole ring, as electron‐deficient C3 position is relatively more susceptible to oxygenation than the C2 position (J. Kim et al, 2019; Leone et al, 2021) (Supporting Information: Figure S3). Among the oxygenases, toluene 4‐monooxygenase from Pseudomonas mendocina KR1 (PmT4MO) can noticeably generate 2‐oxindole from indole, and its F196L mutant enables to direct 74% of the oxygenated indole toward 2‐oxindole formation (McClay et al, 2005).…”

Exploring an anomaly: the synthesis of 7,7′-diazaindirubin through a 7-azaindoxyl intermediate

“…23 Recently, it was shown that cysteine can shi the selectivity of a avin-containing monooxygenase from indigo to indirubin. 24 Herein, an anomaly to the innate selectivity towards indigo under indoxyl-generating conditions is explored for 7-azaindole, 5, which exclusively produces 7,7 0 -diazaindirubin, 6. In this instance, the intermediate is specically 7-azaindoxyl, 7 (Fig.…”