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.