Increased yield of 2-O-a-d-glucopyranosyl-l-ascorbic acid synthesis by a-glucosidase using rational design that regulating the ground state of enzyme and substrate complex

Background: a-Glucosidase (AG) is a bifunctional enzyme, it has a capacity to synthesize 2-O-a-d-glucopyranosyl-l-ascorbic acid (AA-2G) from l-ascorbic acid (L-AA) and low-cost maltose under mild conditions, but it can also hydrolyze AA-2G, which leads to low synthesis efficiency of AA-2G.Main Methods and Major Results: This study introduces a rational molecular design strategy to regulate enzymatic reactions based on inhibiting the formation of ground state of enzyme-substrate complex. Y215 was analyzed as the key amino acid site affecting the affinity of AG to AA-2G and L-AA. For the purpose of reducing the hydrolysis efficiency of AA-2G, the mutant Y215W was obtained by analyzing the molecular docking binding energy and hydrogen bond formation between AG and the substrates. Compared with the wild-type, isothermal titration calorimetry (ITC) results showed that the equilibrium dissociation constant (K-D) of the mutant for AA-2G was doubled; the Michaelis constant (K-m) for AA-2G was reduced by 1.15 times; and the yield of synthetic AA-2G was increased by 39%.Conclusions and Implications: Our work also provides a new reference strategy for the molecular modification of multifunctional enzymes and other enzymes in cascade reactions system.

Automated summary

This study introduces a rational molecular design strategy to regulate enzymatic reactions based on inhibiting the formation of ground state of enzyme-substrate complex. The mutant Y215W was obtained by analyzing the molecular docking binding energy and hydrogen bond formation between AG and the substrates. Compared with the wild-type, the mutant showed improved affinity towards AA-2G and increased synthesis efficiency.