Tuning the Phosphoryl Donor Specificity of Dihydroxyacetone Kinase from ATP to Inorganic Polyphosphate. An Insight from Computational Studies

Abstract

Dihydroxyacetone (DHA) kinase from Citrobacter freundii provides an easy entry for the preparation of DHA phosphate; a very important C3 building block in nature. To modify the phosphoryl donor specificity of this enzyme from ATP to inorganic polyphosphate (poly-P); a directed evolution program has been initiated. In the first cycle of evolution, the native enzyme was subjected to one round of error-prone PCR (EP-PCR) followed directly (without selection) by a round of DNA shuffling. Although the wild-type DHAK did not show activity with poly-P, after screening, sixteen mutant clones showed an activity with poly-phosphate as phosphoryl donor statistically significant. The most active mutant presented a single mutation (Glu526Lys) located in a flexible loop near of the active center. Interestingly, our theoretical studies, based on molecular dynamics simulations and hybrid Quantum Mechanics/Molecular Mechanics (QM/MM) optimizations, suggest that this mutation has an effect on the binding of the poly-P favoring a more adequate position in the active center for the reaction to take place.

Computational study of the phosphoryl donor activity of dihydroxyacetone kinase from ATP to inorganic polyphosphate

“…DHAKs phosphorylate Dha converting it into dihydroxyacetone phosphate (Dha‐P), as shown in Scheme . Dha‐P is a very important intermediate in nature since it is used as phosphoryl donor in several enzyme‐catalyzed aldol reactions by Dha‐P dependent aldolases . Aldolases have been recognized as an indispensable tool for the organic synthesis due to their efficiency to form CC bonds.…”

“…This molecule is overpriced to be used in large‐scale synthesis and is labile at neutral and basic pH values causing the decrease of its effective concentration with time in the enzymatic reaction media . Therefore, an efficient method of Dha‐P preparation is still necessary . In this sense, García‐Junceda and coworkers, on the basis of the recombinant ATP‐dependent DHAK from C. freundii , have elaborated a straightforward multienzyme system for one‐pot CC bond formation catalyzed by DhaP‐dependent aldolases for in situ Dha‐P formation.…”

“…In this sense, García‐Junceda and coworkers, on the basis of the recombinant ATP‐dependent DHAK from C. freundii , have elaborated a straightforward multienzyme system for one‐pot CC bond formation catalyzed by DhaP‐dependent aldolases for in situ Dha‐P formation. In this system, the ATP is regenerated in situ by catalysis of acetate kinase . The fusion protein keep up both kinase and aldolase activity with a very high catalytic efficiency.…”

“…$9/lb can provide ATP equivalents that would cost over $2000/lb separately, while other phosphagens capable of regenerating ATP, such as phosphoenolpyruvate (PEP) and phosphocreatine, cost more than ATP . In addition, a huge quantity of poly‐P is regularly produced as sodium hexametaphosphate (about 13 to 18 residues) for industrial uses such as food additives which also makes poly‐P inexpensive compared to the other phosphoryl donors …”

“…The wild‐type DHAK from C. freundii does not show activity with poly‐P but sixteen mutant clones were found to exhibit certain activity with poly‐P as phosphoryl donor statistically relevant . The most active mutant was based on a single mutation Glu526Lys (E526K), which implies the change of a negative charge for a positive one.…”