Modeling the Structure of Human tRNA-Guanine Transglycosylase in Complex with 7-Methylguanine and Revealing the Factors that Determine the Enzyme Interaction with Inhibitors.

tRNA-guanine transglycosylase, an enzyme catalyzing replacement of guanine with queuine in human tRNA and participating in the translation mechanism, is involved in the development of cancer. However, information on the small-molecule inhibitors that can suppress activity of this enzyme is very limited. Molecular dynamics simulations were used to determine the amino acid residues that provide efficient binding of inhibitors in the active site of tRNA-guanine transglycosylase. It was demonstrated using 7-methylguanine molecule as a probe that the ability of the inhibitor to adopt a charged state in the environment of hydrogen bond acceptors Asp105 and Asp159 plays a key role in complex formation. Formation of the hydrogen bonds and hydrophobic contacts with Gln202, Gly229, Phe109, and Met259 residues are also important. It has been predicted that introduction of the substituents would have a different effect on the ability to inhibit tRNA-guanine transglycosylase, as well as the DNA repair protein poly(ADP-ribose) polymerase 1, which can contribute to the development of more efficient and selective compounds.

Saturation mutagenesis reveals the importance of residues alphaR145 and alphaF146 of penicillin acylase in the synthesis of beta-lactam antibiotics.

Penicillin acylase (PA) from Escherichia coli can catalyze the coupling of an acyl group to penicillin- and cephalosporin-derived beta-lactam nuclei, a conversion that can be used for the industrial synthesis of beta-lactam antibiotics. The modest synthetic properties of the wild-type enzyme make it desirable to engineer improved mutants. Analysis of the crystal structure of PA has shown that residues alphaR145 and alphaF146 undergo extensive repositioning upon binding of large ligands to the active site, suggesting that these residues may be good targets for mutagenesis aimed at improving the catalytic performance of PA. Therefore, site-saturation mutagenesis was performed on both positions and a complete set of all 38 variants was subjected to rapid HPLC screening for improved ampicillin synthesis. Not less than 33 mutants showed improved synthesis, indicating the importance of the mutated residues in PA-catalyzed acyl transfer kinetics. In several mutants at low substrate concentrations, the maximum level of ampicillin production was increased up to 1.5-fold, and the ratio of the synthetic rate over the hydrolytic rate was increased 5-15-fold. Moreover, due to increased tendency of the acyl-enzyme intermediate to react with beta-lactam nucleophile instead of water, mutants alphaR145G, alphaR145S and alphaR145L demonstrated an enhanced synthetic yield over wild-type PA at high substrate concentrations. This was accompanied by an increased conversion of 6-APA to ampicillin as well as a decreased undesirable hydrolysis of the acyl donor. Therefore, these mutants are interesting candidates for the enzymatic production of semi-synthetic beta-lactam antibiotics.

Molecular modeling of different substrate-binding modes and their role in penicillin acylase catalysis.

Molecular modeling was addressed to understand different substrate-binding modes and clarify the role of two positively charged residues of the penicillin G acylase active site - ßR263 and αR145 - in binding of negatively charged substrates. Although the electrostatic contribution to productive substrate binding was dominated by ßR263 rather than αR145, it was found that productive binding was not the only possible mode of substrate placement in the active site. Two extra binding modes - nonproductive and preproductive - were located by means of molecular docking and dynamics with binding affinities comparable with the productive one. A unique feature of nonproductive and preproductive complexes was that the substrate's acyl group did not penetrate the hydrophobic pocket, but occupied a patch on the protein interface spanning from ßR263 to αR145. Nonproductive and preproductive complexes competed with each other and productive binding mode, giving rise to increased apparent substrate binding. Preproductive complex revealed an ability to switch to a productive one during molecular dynamics simulations, and conformational plasticity of the penicillin G acylase active site was shown to be crucial for that. Nonproductive binding observed at molecular modeling corresponded well with experimentally observed substrate inhibition in penicillin acylase catalysis. By combining estimated free energies of substrate binding in each mode, and accounting for two possible conformations of the penicillin G acylase active site (closed and open) quantitative agreement with experimentally measured K(M) values was achieved. Calculated near-attack conformation frequencies from corresponding molecular dynamics simulations were in a quantitative correlation with experimental k(cat) values and demonstrated adequate application of molecular modeling methods.