Abstract
Prenylated indole alkaloids featuring spirooxindole rings possess a 3R or 3S carbon stereocenter, which determines the bioactivities of these compounds. Despite the stereoselective advantages of spirooxindole biosynthesis compared with those of organic synthesis, the biocatalytic mechanism for controlling the 3R or 3S-spirooxindole formation has been elusive. Here, we report an oxygenase/semipinacolase CtdE that specifies the 3S-spirooxindole construction in the biosynthesis of 21R-citrinadin A. High-resolution X-ray crystal structures of CtdE with the substrate and cofactor, together with site-directed mutagenesis and computational studies, illustrate the catalytic mechanisms for the possible β-face epoxidation followed by a regioselective collapse of the epoxide intermediate, which triggers semipinacol rearrangement to form the 3S-spirooxindole. Comparing CtdE with PhqK, which catalyzes the formation of the 3R-spirooxindole, we reveal an evolutionary branch of CtdE in specific 3S spirocyclization. Our study provides deeper insights into the stereoselective catalytic machinery, which is important for the biocatalysis design to synthesize spirooxindole pharmaceuticals.
Highlights
Prenylated indole alkaloids featuring spirooxindole rings possess a 3R or 3S carbon stereocenter, which determines the bioactivities of these compounds
Based on thorough analyses of the high-resolution X-ray crystal structures of CtdE complex containing substrate and cofactor flavin adenine dinucleotide (FAD), together with the site-directed mutagenesis and computational study, we revealed the molecular basis for the stereoselective catalytic mechanism that CtdE exploits for the possible β-facial epoxidation, triggering semipinacol rearrangement to yield 3S-spirooxindole PIAs
The production of 1 was completely abolished in ΔctdQ mutant, which confirmed that the ctd gene cluster is responsible for the biosynthesis of 1 (Fig. 2a)
Summary
Prenylated indole alkaloids featuring spirooxindole rings possess a 3R or 3S carbon stereocenter, which determines the bioactivities of these compounds. NotB has been reported to catalyze the formation of non-spirocyclized notoamides C and D through a presumed indole 2,3-β-epoxide intermediate[22], which does not contain the bicyclo[2.2.2]diazaoctane ring (Fig. 1b). Another FPMO, PhqK, could perform a specific α-face epoxidation on the bicyclo[2.2.2]diazaoctane substrates, triggering semipinacol rearrangement to build up 3R-spirooxindole constructions after the IMDA cyclization[24] (Fig. 1b). Citrinadin A possesses a unique 6/5/5/6/6 pentacyclic ring core with the addition of an N, N-dimethylvaline ester unit and an α, β-epoxy-carbonyl moiety This complex structure with multiple stereocenters has served as a fascinating target in subsequent synthetic studies[27,28,29,30,31]. Unlike paraherquamides[24], notoamides[23], and brevianamides[25], citrinadins do not contain the bicyclo[2.2.2]
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