PHOSPHOENZYMQMMM · Phosphate Processing in Enzymes: Structure, Dynamics and Chemistry

Phosphate Processing in Enzymes: Structure, Dynamics and Chemistry

We have performed a large-scale structural analysis using available crystallographic structures from the Protein Data Bank (PDB). We have identified high-resolution protein complexes where nucleotides with three phosphate groups were present and bound to divalent metal ions. We also identified the catalytic reaction based on the corresponding enzyme commission number (E.C. number) and analysed the metal-ion binding mode in each category. We analysed the coordination geometry in over 700 PDB structures in 15 EC categories. A manuscript based on the results of the PDB analysis, and corresponding QM and QM/MM calculations is currently in preparation and we anticipate that we will submit this shortly for publication. We have established a new collaboration with the University of Cambridge and we have calculated the NMR coupling constants for Cucurbit[n]uril compounds. We have studied the encapsulation and active release of molecular species in a flexible box (oAzoBox4+) by quantum mechanical calculations in collaboration with the group of Prof. Scherman. This work has been published in Agewandte Chemie International Edition and it was selected as the cover of the journal. We have also calculated NMR chemical shifts of complexes that can be used as possible ion sensors. We are evaluating the possibility to design new ion sensors based on the difference in NMR chemical shifts when an ion is bound versus only an unbound ligand is present. We have identified and studied key conserved Arginine residues of dUTPase enzymes and revealed their role in the reaction mechanism. We carried out extensive MD simulations for wild type and two mutant enzymes, for which corresponding x-ray structure was simultaneously determined in our collaborators, Prof. Beata Vertessy’s group (Budeapest Technical University, Hungary). We proposed that R140 is an arginine finger, based on its structural and functional roles. Our experimental collaborators verified this hypothesis, and demonstrated that R140K is nearly inactive. Our QM/MM results agree with the crystallographic structures that both reveal the key role of R140 in structural stabilization of the active site assembly, particularly in positioning the P loop-like motif. We compared the R140 coordination geometry to the NTP phosphate chain with representatives of several enzyme families that carry out one-metal ion catalytic mechanism, and showed the structural identity of the arginine fingers as cluster of arginines contacting the gamma phosphate. Our results identify and characterise the first arginine finger that corresponds to pyrophosphatase enzyme function. Our manuscript based on this work has been published in Journal of American Chemical Society. We have studied several phosphate catalytic enzymes and have results that are either being written up or are important partial results. These other enzymes include topoisomerase, D-Ala-D-Ala ligase and HIV-RT RNase H. Preliminary results on HIV-RT RNase H suggest the existence of two new ligand binding sites in a close proximity to the RNase H active site. We believe that the binding of new inhibitors to these alternative pockets could affect the RNase H activity. We also performed QM/MM calculations, and identified a new proton transfer mechanism via a conserved Histidine, which is also structurally homologous to Histidines in other two-metal ion catalytic systems. A manuscript based on the results is currently in preparation. Some of the Free methods developed in the group were also applied to lipoxygenase (LOX) enzymes. We have studied the first reaction step of human 15-LOX2 enzyme and calculated the reaction free energy profile. For the fist time, we have obtained reaction rates directly from umbrella sampling simulations. This work was published in the Journal of Chemical Theory and Computation.

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