3.3 UDP preference of McSuSy
SuSys, like St SuSy (S. tuberosum L) and SuSyNe(N. europaea ) shows high flexibility for nucleoside diphosphates
in the cleavage reaction.[11, 15] Plant SuSys
preferentially utilizes UDP as an acceptor nucleotide, while bacterial
SuSys prefer ADP. By measuring the kinetic parameters of the enzyme on
the substrate in the sucrose cleavage direction (Table 1), theK m value of Mc SuSy for UDP is 0.13 mM,
indicating that Mc SuSy has a higher affinity for UDP. And it was
difficult to determine the enzyme activity under the same conditions
when ADP was the glycosyl receptor. Homology modeling was carried out
using the crystal structure of At SuSy1 (PDB ID: 3S27) as a
template, which has 55.17% sequence identity with Mc SuSy, and
the complex was obtained by substrate docking using LeDock. The observed
secondary structure of Mc SuSy is very similar to that of theAt SuSy1 monomer (Fig. 2A). Two sequence fragments, residues 333
to 345 and residues 683 to 718, were found in the active site ofMc SuSy, which are highly conserved in plant SuSys (Fig. 2B). To
be specific, the residues 333 to 345 of Mc SuSy (light blue),
corresponding to the residues 300 to 312 of At SuSy1, participate
in the binding of fructose and G336 (G303 in At SuSy1) also
interact with β -phosphate of UDP by forming hydrogen bonds (Figs.
2C and 2D).[22, 24] The residues 683 to 718 ofMc SuSy (pink) corresponding to the residues 648 to 683 ofAt SuSy1 belong to the nucleotide-binding domain, which contains
the “QN” motif, playing a significant role in the nucleotide
preference of SuSy.[13, 24] In particular, the two
amino acids Q683 and N689 (Q648 and N654 in At SuSy1) are highly
conserved in plant SuSys, while in bacteria the residues are highly
variable.[13] For example, R636 and A642 in theN. europaea create a more spacious binding site for the
preference towards the bulkier ADP substrate.[24]As shown in Figs. 2C and 2D, the UDP moieties bind of Mc SuSy are
the same way as At SuSy1, especially in the indicated “QN”
motif, which also implies a similar preference for nucleotide
bases.[22]