3.4 mutation of McSuSy for enhanced activity
Studies have demonstrated that phosphorylation affected the catalytic activities of SuSys in sucrose cleavage, which may increase the apparent affinity of the enzyme for sucrose and UDP to activate the formation of UDP-glucose and fructose from sucrose plus UDP.[33] Three residues including S7, T22, and S31 at the N -terminus of Mc SuSy (Table S2), which were predicted reliably as phosphorylation sites by NetPhos 3.1 Server, were mutated into two different acidic amino acid residues Asp (D) or Glu (E), respectively. Unexpectedly, we found that inclusion bodies of the S7D, S7E, and T22D mutants decreased significantly, and the enzyme activity of the crude extracts declined slightly (Fig. S3 and Fig. 3A). Both S31D and S31E mutants were confirmed to have significantly increased enzyme activity in crude extracts (more than 50% compared with wild-type Mc SuSy) and had little effect on the soluble expression. After purification, the kinetic parameter of S31D was determined (Table 1). The K m values of S31D were 70.18 mM and 0.09 mM for sucrose and UDP, respectively, indicating a drop of more than 20% and an increased apparent affinity for subtracts compared with the wild type of Mc SuSy (Figs. S4 and S5).
Then seven residues surrounding the nucleobase ring of UDP in the “QN” motif,[13, 22] were evaluated by consensus analysis based on sequence alignment of the identified SuSys, to further improve the activity of Mc SuSy (Fig. 2B). Only K684 and N685 having the top three or two highest probabilities of alternative residues were chosen for mutagenesis. Other residues in the “QN” motif were very conservative. Three single-site mutants K684M, K684T, and N685D were generated, and the enzyme activities of the crude extracts were measured. The enzyme activities of the mutants K684T and N685D were 126.4% and 149.8% of those of the wild type, respectively (Fig. 4B). Subsequently, the mutant N685D was used as the template to overlie the other two mutations to obtain the multi-site mutants. Compared to the wild type, the crude enzyme activities of three multi-site mutants, that was, K684T/K685D, S31D/K685D, and S31D/684T/685D, were increased by 60%, among which the mutant S31D/684T/685D named Mc SuSym was the highest. Interestingly, Mc SuSym exhibited comparable activity as the mutant S31D. Moreover, enzymatic kinetic assays (Table 1) showed that Mc SuSym is higher than the wild type for sucrose catalytic efficiency (K cat/K m).