Triterpenoids are a kind of important natural products of plants, which have attracted extensive attention due to their unique pharmacological activities。However, due to the hydrophobic carbon ring structure in the molecule, triterpenes generally have the problem of low solubility and poor bioavailability。The water solubility of triterpenes can be significantly improved by glycosylation modification, so triterpenes mostly exist in the form of glycosides。Among them, the straight chain triosaccharide modification is an important type of glycosylation, which plays an important role in exerting the unique pharmacological activity of triterpenoids。Compared with chemical synthesis, enzymatic synthesis of triterpene glycosides has the advantages of mild reaction conditions, directional catalysis and environmental friendliness, especially in the synthesis of triterpene glycosides with complex polysaccharide group modification, enzymatic synthesis can avoid the complicated group protection and deprotection process in chemical synthesis。uridine diphosphate glycosyltransferases，UGTs is the main biocatalyst to catalyze the glycosylation of natural products，In the UGTs that have been reported so far，Most of them only catalyze the synthesis of triterpenoid monoglycosides and diglycosides，The number of UGTs with linear triosaccharide modification catalytic activity is very small，The enzymatic synthesis of triterpene straight chain triglycosides was limited。In addition, the catalytic function characteristics of this type of UGTs and the molecular mechanism of substrate specificity are not clear。Aiming at the above problems，In this study, bioinformatics analysis and in vitro enzymatic characterization system were used to explore the UGTs that can catalyze the synthesis of triterpene straight chain triglycosides，The structural characteristics of the catalytic substrates of UGTs were also revealed，The molecular mechanism of substrate specificity was elucidated by means of ancestral enzyme sequence reconstruction, amino acid mutation and computer simulation。相关研究成果以“Engineering the Substrate Specificity of UDP-Glycosyltransferases for Synthesizing Triterpenoid Glycosides with a Linear Trisaccharide as Aided by Ancestral Sequence Reconstruction”为题目在国际知名期刊Angewante Chemie International Edition上发表（DOI: /10.1002/anie.202409867）。First author of the paper, Professor Li Chun from Tsinghua University and Professor Feng Xudong from the School of Chemistry and Chemical Engineering at Beijing Institute of Technology are co-corresponding authors of the paper。

In this study, 7 UGT91H subfamily enzymes were systematically characterized. The results showed that 7 UGTs had catalytic activity on 14 triterpenes, and they specifically modified a sugar group in their C2 "-OH, and successfully synthesized 26 triterpene straight chain triglycosides。The structural characteristics of glycosyl-receptor substrates catalyzed by 7 UGT91H subfamily enzymes were revealed: they could only catalyze triterpenoids modified by C3-OH and the first glucuronyl or glucuronyl, while they had no catalytic activity for triterpenoid aglycones or triterpenoid monoglycosides。In addition, the 7 UGTs showed strict specificity to UDP-glycosyl donors, the highest preference to UDp-rhamnose (UDP-Rha), followed by UDP-xylose (UDP-Xyl), and no catalytic activity to other UDP-glycosyl donors。

The ancestor enzyme UGT91H_A1 of the UGT91H subfamily was obtained by using the ancestral enzyme sequence reconstruction technique, which showed a wider substrate spectrum and higher activity while retaining the function of the modern enzymes of the UGT91H subfamily。UGT91H_A1 can catalyze 17 triterpenes to modify a single glycosyl receptor。In addition to UDP-Rha and UDP-Xyl, UGT91H_A1 also has the activity of efficient utilization of UDP-glucose (UDP-Glc)。Finally, 33 straight chain triterpene triglycosides were synthesized by UGT91H_A1。

The non-conserved RTAS Loop of UGT91H_A1 has an important effect on the substrate specificity of UGT91H subfamily enzymes。The RTAS Loop was replaced with the corresponding region of UGT91H8 and UGT91H7, and the mutants UGT91H_6mu and UGT91H7M were obtained, respectively, which had the activity of catalytic glycosylation of the new substrate α-ivoside (17)。Molecular dynamics simulations and quantum mechanical calculations show that the RTAS Loop regulates the substrate specificity of enzymes by influencing the interaction and binding energy of enzymes and substrates。

Addressing the high cost of glycosyl donor UDP-Rha，A three-enzyme cascade system including sucrose synthase Gu SUS1-Δ9, UDP-Rha synthase At RHM2 and bifunctional rhamnose-synthase ATNRS /ER was constructed，The UDP-Rha was synthesized from cheap sucrose。The cost of synthesis of 3-O-GlcA-Gal-Rha (2a) and 3-O-Ara-Rha-Rha (17a) was reduced by the four-enzyme system coupled with UGT91H_A1 (step method)，The substrate conversion rate reached 80 respectively.1 ± 0.3%和61.3 ± 4.1%。

The sequence of UGT91H subfamily enzymes characterized above was used as the probe，A rapid enzyme mining method based on evolutionary analysis was established，The genome of 8 species of legume in NCBI database was analyzed and mined systematically，A total of 23 candidate enzymes were obtained，Finally, 19 enzymes were identified as UGT91H subfamily，The efficiency and accuracy of enzyme mining are greatly improved。PNY09112 was selected.1. GAU40116.1 and PNX78912.The functional characterization of 1 showed that the triterpenoid compound C2 "-OH could specifically modify one molecule of rhamnosyl or xylosyl。Also, GAU40116.1 and PNY09112.1 also has a weak new activity against UDP-Gal。

In this study, the catalytic function of UGT91H subfamily enzymes was systematically explored, and the substrate profile of UGT91H subfamily enzymes was expanded by using ancestral enzyme reconstruction technology, and 23 novel triterpene straight-chain triglycosides were synthesized by enzymatic method。The RTAS Loop, a key region affecting the substrate specificity of UGT91H_A1, was identified, and the substrate specificity of UGT91H subfamily enzymes was regulated by mutating the corresponding region of RTAS Loop。A rapid mining method for UGT91H subfamily enzymes based on evolutionary analysis was also developed。This study provides a new idea and method for further developing more enzymatic glycosylation modification types of triterpenoids。