Structural basis of the stereoselective formation of the spirooxindole ring in the biosynthesis of citrinadins

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.

Discovery of Semi-Pinacolases from the Epoxide Hydrolase Family during Efficient Assembly of a Fungal Polyketide

“…However, few SPases have been identified in limited enzyme families, and all discovered SPases showed catalysis of the SPR on alkaloids after oxidations (epoxidation or hydroxylation). One SPase (FtmG) catalyzes type II SPR with an allylic alcohol as the substrates and two SPases (BvnE and AuaG) catalyze type IV SPR. , However, other SPases (NotB, PhqK, FqzB, CtdE, NotI/NotI′, CyaH) ,,,− are oxidases and catalyze epoxidation-coupled type III SPR in an R3 → C3 2,3-migration manner, to shift the substituent groups and confer the structural complexity, such as spirocycles (Schemes S1–S3).…”

“…To date, several SPases have been identified in limited families only with alkaloids as the substrates (Scheme a). Most SPases are bifunctional enzymes belonging to flavin-dependent monooxygenases (FMOs) (NotI/NotI′, NotB, PhqK, FqzB, AuaG, CtdE) − and the P450 monooxygenase (CyaH) to catalyze epoxidation on indole/quinolone alkaloids to produce epoxy amines and subsequent SPR. Other SPases include the NTF2-like superfamily enzyme (BvnE) and a bifunctional cytochrome P450 (FtmG) to catalyze SPR after hydroxylation on the indole of alkaloids.…”

Rational design of allosteric switchable catalysts

“…Recently, versatile molecular scaffolds were utilized to afford exceptional examples aiming at natural enzymes' expertise: facilitating the formation of enzymesubstrate complex and reducing the activation energy barrier of catalytic reactions. Essential knowledge and critical factors (e.g., hydrogen bonds, electrostatic potential, van der Waals' forces, coordination bonds) that govern the enzyme activity have been explored by studying different models such as small organic molecules, [14][15][16][17][18][19][20] polymers, [21][22][23][24][25][26][27][28][29] supramolecular complexes, [30][31][32][33][34][35][36][37] natural proteins, [38][39][40][41][42][43][44] and protein assemblies. [45][46][47][48][49][50][51][52] These multidisciplinary works have spawned many successful bioinspired enzymes, some of which even showed high efficiencies comparable to their natural counterparts.…”

Electrochemical oxidative rearrangement of tetrahydro-β-carbolines in a zero-gap flow cell

“…Oxidative rearrangement of tetrahydro-b-carbolines (THbCs) 1 (Fig. 1) was proposed as an enzyme-mediated reaction [2][3][4] responsible for biosynthesis of many biologically active spiro [indolizidine-1,3-oxindole] (i.e., spirooxindoles) natural products such as mitraphylline, rhynchophylline, (iso)corynoxeine, corynoxine, corynoxine B, spirotryprostatins A and B, and alstonisine (Fig. 1).…”