Candida Antarctica lipase B (CALB) is a well-known enzyme, especially because of its promiscuous activity. Due to its properties, CALB was widely used as a benchmark for designing new catalysts for important organic reactions. The active site of CALB is very similar to that of soluble epoxide hydrolase (sEH) formed by a nucleophile-histidine-acid catalytic triad and an oxyanion hole typical for molecular structures derived from processes of α/β hydrolases. In this work we are exploring these similarities and proposing a Ser105Asp variant of CALB as a new catalyst for epoxide hydrolysis. In particular, the hydrolysis of the trans-diphenylpropene oxide (t-DPPO) is studied by means of quantum cluster models mimicking the active site of both enzymes. Our results, based on semi-empirical and DFT calculations, suggest that mutant Ser105Asp CALB is a good protein scaffold to be used for the bio-synthesis of chiral compounds.
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.
Protein kinases, representing one of the largest protein family involved in almost all aspects of cell life, have become one of the most important targets for the development of new drugs to be used in, for instance, cancer treatments. In this paper an exhaustive theoretical study of the phosphoryl transfer reaction from adenosine triphosphate (ATP) to dihydroxyacetone (Dha) catalyzed by DhaK from Escherichia coli (E. coli) is reported. Two different mechanisms, previously proposed for the phosphoryl transfer from ATP to the hydroxyl side chain of specific serine, threonine or tyrosine residues, have been explored based on the generation of free energy surfaces (FES) computed with hybrid QM/MM potentials. The results suggest that the substrate-assisted phosphoryl and proton-transfer mechanism is kinetically more favorable than the mechanism where an aspartate would be activating the Dha. Although the details of the mechanisms appear to be dramatically dependent on the level of theory employed in the calculations (PM3/MM, B3LYP:PM3/MM or B3LYP/MM), the transition states (TSs) for the phosphoryl transfer step appear to be described as a concerted step with different degrees of synchronicity in the breaking and forming bonds process in both explored mechanisms. Residues of the active site belonging to different subunits of the protein such as Gly78B, Thr79A, Ser80A, Arg178B and one Mg 2+ cation would be stabilizing the transferred phosphate in the TS. Asp109A would have a structural role by posing the Dha and other residues of the active site in the proper orientation. The information derived from our calculations not only reveals the role of the enzyme and the particular residues of its active site, but it can assist in the rationally design of new more specific inhibitors.3
Phosphoryl transfer reactions are ubiquitous in biology, being involved in processes ranging from energy and signal transduction to the replication genetic material. Dihydroxyacetone phosphate (Dha--P), an intermediate of the synthesis of pyruvate and a very important building block in nature, can be generated converting free dihydroxyacetone (Dha) through the action of Dihydroxyacetone kinase enzyme. In this paper the reference uncatalyzed reaction in solution has been studied in order to define the foundations of the chemical reaction and to determine the most adequate computational method to describe this electronically complex reaction. In particular, the phosphorylation reaction mechanism between adenosine triphosphate (ATP) and Dha in aqueous solution has been studied by means of quantum mechanics/molecular mechanics (QM/MM) Molecular Dynamics (MD) simulations with the QM subset of atoms described with semi--empirical and DFT methods. The results appear to be strongly dependent on the level of calculation, which will have to be taken into account for future studies of the reaction catalyzed by enzymes. In particular, while PM3/MM renders lower free energy barriers and a less endergonic process than AM1d/MM and PM6/MM methods. Nevertheless, the concerted pathway was not located with the former combination of potentials.
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