Local Electric Field Modulated Reactivity of Pseudomonas aeruginosa Acid Phosphatase for Enhancing Phosphorylation of l-Ascorbic Acid

Acid phosphatases (APases) are attractive enzymes for catalyzing large-scale industrial phosphorylation reactions owing to their capacity of utilizing cheap phosphate donors as phosphate sources as well as their broad substrate spectrum. However, APases exhibit strong hydrolytic activity that usually overwhelms the needed phosphorylation reaction. In the present study, we have solved the crystal structure of APase from Pseudomonas aeruginosa (PaAPase) and unraveled the mechanism of PaAPase-catalyzed l-ascorbic acid phosphorylation using multiscale computational studies. In addition, we have engineered the charged residues near the active site to investigate the local electric field effects on modulating the competition between hydrolysis and phosphorylation in PaAPase. In the optimal variant of Q6 containing Asp135 → Arg135 mutation, the corresponding phosphorylation/hydrolysis ratios have increased by 2.9-fold compared with those in the wild-type enzyme. In particular, our simulations show that the local electric field of Q6 could remarkably inhibit the hydrolysis of the phospho-His171 intermediate while having relatively minor effects on the overall phosphorylation reactions. Such an introduced local electric field shifts the phosphorylation/hydrolysis balance in favor of phosphorylation reaction. Our combined experiments and theories demonstrate that protein engineering focusing on local electric field optimization is a practical strategy for modulating enzymatic reactivity.