Discovering a uniform functional trade-off of the CBC-type 2,3-oxidosqualene cyclases and deciphering its chemical logic.

Many functionally promiscuous plant 2,3-oxidosqualene cyclases (OSCs) have been found, but complete functional reshaping is rarely reported. In this study, we have identified two new plant OSCs: a unique protostadienol synthase (AoPDS) and a common cycloartenol synthase (AoCAS) from Alisma orientale (Sam.) Juzep. Multiscale simulations and mutagenesis experiments revealed that threonine-727 is an essential residue responsible for protosta-13 (17),24-dienol biosynthesis in AoPDS and that the F726T mutant completely reshapes the native function of AoCAS into a PDS function to yield almost exclusively protosta-13 (17),24-dienol. Unexpectedly, various native functions were uniformly reshaped into a PDS function by introducing the phenylalanine → threonine substitution at this conserved position in other plant and non-plant chair-boat-chair-type OSCs. Further computational modeling elaborated the trade-off mechanisms of the phenylalanine → threonine substitution that leads to the PDS activity. This study demonstrates a general strategy for functional reshaping by using a plastic residue based on the decipherment of the catalytic mechanism.

Oxidosqualene Cyclases Involved in the Biosynthesis of Diverse Triterpenes in Camellia sasanqua.

Camellia sasanqua is an important economic plant that is rich in lipophilic triterpenols with pharmacological activities including antiallergic, anti-inflammatory, and anticancer activities. However, the key enzymes related to triterpene biosynthesis have seldom been studied in C. sasanqua. Oxidosqualene cyclases (OSCs) are the rate-limiting enzymes related to triterpene biosynthesis. In this study, seven putative OSC genes (CsOSC1-7) were mined from the C. sasanqua transcriptome. Six CsOSCs were characterized for the biosynthesis of diverse triterpene skeletons, including α-amyrin, ß-amyrin, δ-amyrin, dammarenediol-II, ψ-taraxasterol, taraxasterol, and cycloartenol by the heterologous expression system. CsOSC3 was a multiple functional α-amyrin synthase. Three key residues, Trp260, Tyr262, and Phe415, are critical to the catalytic performance of CsOSC3 judging from the results of molecular docking and site-directed mutagenesis. These findings provide important insights into the biosynthesis pathway of triterpenes in C. sasanqua.

Exploring the catalytic function and active sites of a novel C-glycosyltransferase from Anemarrhena asphodeloides.

Anemarrhena asphodeloides is an immensely popular medicinal herb in China, which contains an abundant of mangiferin. As an important bioactive xanthone C-glycoside, mangiferin possesses a variety of pharmacological activities and is derived from the cyclization reaction of a benzophenone C-glycoside (maclurin). Biosynthetically, C-glycosyltransferases are critical for the formation of benzophenone C-glycosides. However, the benzophenone C-glycosyltransferases from Anemarrhena asphodeloides have not been discovered. Herein, a promiscuous C-glycosyltransferase (AaCGT) was identified from Anemarrhena asphodeloides. It was able to catalyze efficiently mono-C-glycosylation of benzophenone, together with di-C-glycosylation of dihydrochalcone. It also exhibited the weak O-glycosylation or potent S-glycosylation capacities toward 12 other types of flavonoid scaffolds and a simple aromatic compound with -SH group. Homology modeling and mutagenesis experiments revealed that the glycosylation reaction of AaCGT was initiated by the conserved residue H23 as the catalytic base. Three critical residues H356, W359 and D380 were involved in the recognition of sugar donor through hydrogen-bonding interactions. In particular, the double mutant of F94W/L378M led to an unexpected enzymatic conversion of mono-C- to di-C-glycosylation. This study highlights the important value of AaCGT as a potential biocatalyst for efficiently synthesizing high-value C-glycosides.