4 DISCUSSION
In the present study, the prokaryotic An SuSy and Mr SuSy were used as templates for sequence collection, and the homology, as well as the active site of the reported SuSys, were also considered in the sequence screening process. Particularly, the sequences that have the conserved residues contributing to UDPG binding, corresponding to Q648, N654, and E683 in At SuSy1 remained.[11, 24] The phylogenetic tree shows the classification and evolutionary relationship of three selected sequences (Mc SuSy, Cb SuSy1, and Cb SuSy2) from the algae and several other characterized SuSys from plants and bacteria (Fig. S6). They are close to those from plants, falling in the Eukaryotic group, and share the common conserved active site residues in retaining GT-B glycosyltransferases (Table S3), which was known from the multiple sequence alignment. What we focused on was Mc SuSy, which was heterologously expressed in E. coli in a more soluble form and with higher activity than Cb SuSy1 and Cb SuSy2. Lower pH values are known to promote the cleavage reaction of SuSys, yielding NDP-glucose and fructose, and with the increasing of the pH, NDP-glucose synthesis is disfavored.[2-4] While theMc SuSy displayed the highest activity at pH 7.0 in sucrose degradation (Fig. 1B), which is different from other sources of SuSys preferring to hydrolyze sucrose at acidic pH. And it is suitable to apply in SuSy-GT cascade reactions coupling with Leloir GT having the optimal neutral pH. With the residual activity of above 80% after 15 min of incubation at 42°C with sucrose, the efficient recycling of UDPG may be realized by appropriately increasing the temperature of the catalytic reaction.
In addition, the plant SuSy is a known phosphoserine-containing enzyme.[36] One distinctive characteristic feature of SuSys is that phosphorylation of the N -terminus at the major phosphorylation site in plants contributes to the fine-tuning of enzyme activity and may be responsible for changes in membrane binding.[36, 37] In contrast, Interestingly, theN -terminal sequence alignment of prokaryotic SuSys shows that a highly conserved motif was found in cyanobacteria SuSys as a putative phosphoacceptor, but for non-cyanobacteria SuSys, there is no definite motif to distinguish.[4] Previous studies have shown that phosphorylation or introducing the negative charge at the N -terminal phosphorylation site of plant SuSys, such as at S15 of Zm SuSy and S11 ofGm SuSy and St SuSy1, has affected their catalytic activities in sucrose cleavage.[36-38] In theN -terminal sequence alignment of four SuSys involvingMc SuSy, Gm SuSy, StS uSy1, and Zm SuSy, the reported phosphorylation site is conserved (Fig. S7) in Mc SuSy (S31), which is identical to the predicted results obtained from NetPhos 3.1 Server (Table S2). S31D mutation of Mc SuSy showed a nearly 1.2-fold increase in the enzyme activity, which suggest that induction of the negative charge at S31, like phosphorylation, may affect theN -terminal conformation and the interactions between adjacent region, thus stimulating the catalytic activity ofMc SuSy.[22] Low K mvalues for UDP are beneficial for in vitro recycling of UDPG in SuSy-GT coupled systems due to favored sucrose cleavage, and the product can be synthesized with endogenesis UDP. The K m ofMc SuSy for UDP (0.13 mM) is almost comparable to that of plant SuSys (Table S4) and 1.5 times higher than the S31D mutation. The affinity for sucrose, indicated by the K m value, was much worse than that of plants, although improved after mutation, which implies that Mc SuSy would not be inhibited by high concentrations of sucrose. When the residues in the “QN” motif were mutated, the affinity of Mc SuSym to UDP reduced, compared to those of the wild type and S31D mutant of Mc SuSy (Table 1), which may be caused by changing the interaction of residues binding with UDP.
Ginsenosides are the major pharmacological active compounds in traditional Chinese medicine ginseng. As a promising candidate drug for cancer prevention and treatment, PPD-type ginsenoside Rh2 has gradually aroused great interest in the medicinal and healthcare industries.[34, 35] However, the content of ginsenoside Rh2 in red ginseng is relatively low (0.0001% – 0.0003% in dried ginseng roots).[39] Due to the long cultivation time of ginseng, the complex extraction and purification process of bioactive compounds, at present, the synthesis of Rh2 mainly depends on biological deglycosylation of PPD-type ginsenosides (such as ginsenoside Rb1, Rb2 and Rc).[40] Moreover, ginsenoside Rh2 also can be obtained by heterologous de­­­-novo synthesis through the construction of a synthesis pathway in a yeast cell factory.[41] However, some issues, such as the toxicity of ginsenosides to host cells, the low content of PPD-type ginsenosides in ginseng, and the poor efficiency of enzymatic hydrolysis, still limit Rh2 production. UDP-glycosyltransferases with regiospecificity, such as PgUGT74AE2, UGTPg45 from P. ginseng , and UGT73C5 from A. thaliana ,[42, 43] are responsible for the PPD-type and PPT-type ginsenoside (Rh2, CK, Rh2, F2, and Rh1) synthesis, providing diverse options of GTs for constructing the cost-effective SuSy-GT cascade reactions. Up to now, Rh2 has been successfully synthesized from PPD by UGT73C5 from A. thalianacoupling At SuSy1, and Bs-YjiC from Bacillus subtiliscoupling At SuSy1.[42, 44] In such reactions, a high concentration of DMSO was used as a cosolvent of PPD, the high reaction efficiency was obtained by constantly adding fresh enzyme solutions. Thus, the stability of plant-derived UGT and SuSy has a vital impact on the application and amplification of biotransformation of PPD to produce Rh2. For Mc SuSy, the optimum temperature is 60 °C, and its enzyme activity may be well maintained in presence of sucrose, indicating higher thermostability than those of plant origin. At the same time, the increasing temperature usually improves the solubility of substances and the viscosity of the solution. Therefore, higher temperature conditions are more conducive to promoting the transformation of substrates with high concentrations, especially for those with low solubility.
In brief, benefiting from the UDP preference and the inherently better thermostability, Mc SuSy may be able to work as a competitive rival of plant and bacteria SuSys for in situ regeneration of UDPG to promote the glycosylation catalyzed by a variety of Leloir GTs.