Characterization of a Malabaricane-Type Triterpene Synthase from Astragalus membranaceus and Enzymatic Synthesis of Astramalabaricosides

Abstract

Triterpenoids are a large and medicinally important group of natural products with a wide range of biological and pharmacological effects. Among them, malabaricane-type triterpenoids are a rare group of terpenoids with a 6,6,5-tricyclic ring system, and a few malabaricane triterpene synthases have been characterized to date. Here, an arabidiol synthase AmAS for the formation of the malabaricane-type 6,6,5-tricyclic triterpenoid skeleton in astramalabaricosides biosynthesis was characterized from Astragalus membranaceus. Multiple sequence alignment, site-directed mutagenesis, and molecular docking of AmAS reveal that residues Q256 and Y258 are essential for AmAS activity, and the triad motif IIH725-727 was the critical residue necessary for its product specificity. Mutation of IIH725-727 with VFN led to the formation of seven tricyclic, tetracyclic, and pentacyclic triterpenoids (1–7). Glycosylation of malabaricane-type triterpenoids in the biosynthesis of astramalabaricosides was also explored. Three triterpenoids (1, 5, and 6) displayed potent inhibitory effects against influenza A virus in vitro. These findings provide insights into malabaricane-type triterpenoids biosynthesis in A. membranaceus and access to diverse bioactive triterpenoids for drug discovery.

Rhabdastrellosides A and B: Two New Isomalabaricane Glycosides from the Marine Sponge Rhabdastrella globostellata, and Their Cytotoxic and Cytoprotective Effects

“…Site-directed mutagenesis of the key residue IIH725-727 with VFN led to seven triterpenoidal products, including arabidiol (3) and compound 4, which are partially malabaricane analogues of the aglycon parts in 1 and 2 (Figure 8). Further experimental glycosylation of triterpenoidal substrate 3 with plant glycosyltransferases from A. membranaceus led to the products containing 3-O-mono-and disaccharide chains [27]. Based on the structural similarity between the investigated malabaricanes and new isomalabaricanes 1 and 2, we may suggest that glycosylation is a prior or parallel step to oxidation at C-12 in the biosynthesis of both types of triterpenoids.…”

“…Site-directed mutagenesis of the key residue IIH725-727 with VFN led to seven triterpenoidal products, including arabidiol (3) and compound 4, which are partially malabaricane analogues of the aglycon parts in 1 and 2 (Figure 8). Further experimental glycosylation of triterpenoidal substrate 3 with plant glycosyltransferases from A. membranaceus led to the products containing 3-O-mono-and disaccharide chains [27].…”

“…The acetylated 2-octylglycosides were purified by silica gel (2 × 0.5 cm) flash chromatography in 100% MeOH and then analyzed by GC-MS using the corresponding authentic samples prepared by the same procedure (Figures S23-S27) [30]. The retention times (t R ) and EIMS spectra showed for the components of the hydrolysate corresponded to those of the authentic samples and were as follows: D-glucose: t R (9), 215 (7), 200 (55), 182 (9), 169 (58), 157 (100), 140 (57), 127 (18), 115 (96), 98 (95), 81 (32), 71 (25), 57 (34); t R 28.80 min, m/z (%): 354 (2), 331 (36), 289 (3), 271 (5), 242 (42), 229 (8), 200 (45), 182 (9), 169 (100), 157 (89), 140 (46), 127 (21), 115 (87), 98 (74), 81 (24), 71 (25), 57 (33) and t R 29.13 min, m/z (%):354 (2), 331 (100), 315 (9), 289 (2), 259 (2), 242 (11), 227 (9), 215 (5), 203 (16), 183 (12), 169 (71), 157 (27), 143 (60), 127 (23), 115 (46), 98 (62), 81 (25), 71 (36), 57 (40); N-acetyl-D-glucosamine: t R 31.51 min, m/z (%): 355 (4), 330 (8), 318 (3), 288 (4), 259 (5), 241 (52), 226 (10), 210 (10), 199 (38), 181 (40), 168 (20), 156 (66), 139 (72), 126 (22), 114 (100), 96 (49), 84 (27), 72 (20), 57 (18).…”