Conformational Changes and ATP Hydrolysis in Zika Helicase: The Molecular Basis of a Biomolecular Motor Unveiled by Multiscale Simulations.

We computationally study the Zika NS3 helicase, a biological motor, using ATP hydrolysis energy for nucleic acid remodeling. Through molecular mechanics and hybrid quantum mechanics/molecular mechanics simulations, we explore the conformational landscape of motif V, a conserved loop connecting the active sites for ATP hydrolysis and nucleic acid binding. ATP hydrolysis, initiated by a meta-phosphate group formation, involves the nucleophilic attack of a water molecule activated by Glu286 proton abstraction. Motif V hydrogen bonds to this water via the Gly415 backbone NH group, assisting hydrolysis. Posthydrolysis, free energy is released when the inorganic phosphate moves away from the coordination shell of the magnesium ion, inducing a significant shift in the conformational landscape of motif V to establish a hydrogen bond between the Gly415 NH group and Glu285. According to our simulations, the Zika NS3 helicase acts as a ratchet biological motor with motif V transitions steered by Gly415's γ-phosphate sensing in the ATPase site.

Elucidation of the Active Form and Reaction Mechanism in Human Asparaginase Type III Using Multiscale Simulations.

l-asparaginases catalyze the asparagine hydrolysis to aspartate. These enzymes play an important role in the treatment of acute lymphoblastic leukemia because these cells are unable to produce their own asparagine. Due to the immunogenic response and various side effects of enzymes of bacterial origin, many attempts have been made to replace these enzymes with mammalian enzymes such as human asparaginase type III (hASNaseIII). This study investigates the reaction mechanism of hASNaseIII through molecular dynamics simulations, quantum mechanics/molecular mechanics methods, and free energy calculations. Our simulations reveal that the dimeric form of the enzyme plays a vital role in stabilizing the substrate in the active site, despite the active site residues coming from a single protomer. Protomer-protomer interactions are essential to keep the enzyme in an active conformation. Our study of the reaction mechanism indicates that the self-cleavage process that generates an N-terminal residue (Thr168) is required to activate the enzyme. This residue acts as the nucleophile, attacking the electrophilic carbon of the substrate after a proton transfer from its hydroxyl group to the N-terminal amino group. The reaction mechanism proceeds with the formation of an acyl-enzyme complex and its hydrolysis, which turns out to be the rate-determining step. Our proposal of the enzymatic mechanism sheds light on the role of different active site residues and rationalizes the studies on mutations. The insights provided here about hASNaseIII activity could contribute to the comprehension of the disparities among different ASNases and might even guide the design of new variants with improved properties for acute lymphoblastic leukemia treatment.

Mechanistic study of the biosynthesis of R-phenylacetylcarbinol by acetohydroxyacid synthase enzyme using hybrid quantum mechanics/molecular mechanics simulations.

The biosynthesis of R-phenylacetylcarbinol (R-PAC) by the acetohydroxy acid synthase, (AHAS) is addressed by molecular dynamics simulations (MD), hybrid quantum mechanics/molecular mechanics (QM/MM), and QM/MM free energy calculations. The results show the reaction starts with the nucleophilic attack of the C2α atom of the HEThDP intermediate on the Cß atom of the carbonyl group of benzaldehyde substrate via the formation of a transition state (TS1) with the HEThDP intermediate under 4'-aminopyrimidium (APH+) form. The calculated activation free energy for this step is 17.4 kcal mol-1 at 27 °C. From this point, the reaction continues with the abstraction of Hß atom of the HEThDP intermediate by the Oß atom of benzaldehyde to form the intermediate I. The reaction is completed with the cleavage of the bond C2α-C2 to form the product R-PAC and to regenerate the ylide intermediate under the APH+ form, allowing in this way to reinitiate to the catalytic cycle once more. The calculated activation barrier for this last step is 15.9 kcal mol-1 at 27 °C.

Mechanistic study of the biosynthesis of R-phenylcarbinol by acetohydroxyacid synthase enzyme using hybrid quantum mechanics/molecular mechanics simulations.

The biosynthesis of R-phenylacetylcarbinol (R-PAC) by the acetohydroxy acid synthase, (AHAS) is addressed by molecular dynamics simulations (MD), hybrid quantum mechanics/molecular mechanics (QM/MM), and QM/MM free energy calculations. The results show the reaction starts with the nucleophilic attack of the C2α atom of the HEThDP intermediate on the Cß atom of the carbonyl group of benzaldehyde substrate via the formation of a transition state (TS1) with the HEThDP intermediate under 4'-aminopyrimidium (APH+) form. The calculated activation free energy for this step is 17.4kcal mol-1 at 27 °C. From this point, the reaction continues with the abstraction of Hß atom of the HEThDP intermediate by the Oß atom of benzaldehyde to form the intermediate I. The reaction is completed with the cleavage of the bond C2α-C2 to form the product R-PAC and to regenerate the ylide intermediate under the APH+ form, allowing in this way to reinitiate to the catalytic cycle once more. The calculated activation barrier for this last step is 15.9kcal mol-1 at 27 °C.

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

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.