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.
Highlights
Dihydroxyacetone (DHA) kinase from Citrobacter freundii (C. freundii) provides an easy entry for the preparation of DHA phosphate, a very important C3 building block in nature since it is used as phosphoryl donor in several enzyme-catalyzed aldol reactions [1]
Dihydroxyacetone phosphate (DHAP)-dependent aldolases are among the most important biocatalysts for C–C bond formation [5,6,7,8,9,10]. Their major synthetic advantage is that the stereochemistry of the two newly formed stereogenic centers is controlled by the enzymes and, the four DHAP-dependent aldolases are stereocomplementary; so, from two given substrates, it is possible to obtain the four diastereoisomers
Plates were centrifuged at 3000 rpm for 5 min and the cell free extracts (CFE) were transferred to new 96-well plates to carry out the activity assay
Summary
Dihydroxyacetone (DHA) kinase from Citrobacter freundii (C. freundii) provides an easy entry for the preparation of DHA phosphate, a very important C3 building block in nature since it is used as phosphoryl donor in several enzyme-catalyzed aldol reactions [1]. Dihydroxyacetone phosphate (DHAP)-dependent aldolases are among the most important biocatalysts for C–C bond formation [5,6,7,8,9,10] Their major synthetic advantage is that the stereochemistry of the two newly formed stereogenic centers is controlled by the enzymes and, the four DHAP-dependent aldolases are stereocomplementary; so, from two given substrates, it is possible to obtain the four diastereoisomers. Besides the phosphorylation of DHA, DHAK is able to catalyze, in the same active center, the cyclization of flavin adenine dinucleotide (FAD) to cyclic flavin mononucleotide (FMN) This catalytic promiscuity is modulated by the divalent cation that forms the complex with the phosphorylated substrate. A proposal of the origin of the activity will be based on the simulations
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