Tuning the phosphoryl donor specificity of dihydroxyacetone kinase from ATP to inorganic polyphosphate. An insight from computational studies

Abstract

In this work, we studied Dihydroxyacetone (DHA) kinase from Citrobacter freundii, an enzyme that provides an efficient pathway for the preparation of DHA phosphate, a crucial C3 building block in nature. To modify the phosphoryl donor specificity of this enzyme from ATP to inorganic polyphosphate (poly-P), we initiated a directed evolution program. In the first cycle of evolution, the native enzyme was subjected to one round of error-prone PCR (EP-PCR) followed directly by DNA shuffling, without intermediate selection. Although the wild-type DHAK showed no activity with poly-P, screening revealed sixteen mutant clones with statistically significant activity using poly-P as the phosphoryl donor. The most active mutant featured a single mutation (Glu526Lys) located in a flexible loop near the active center. Through theoretical studies based on molecular dynamics simulations and hybrid Quantum Mechanics/Molecular Mechanics (QM/MM) optimizations, we demonstrated that this mutation facilitates the binding of poly-P, positioning it more favorably in the active center for the reaction to occur.