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).