Swarnalatha Y. Reddy

Mechanism of methanol oxidation by quinoprotein methanol dehydrogenase

At neutral pH, oxidation of CH3OH → CH2O by an o-quinone requires general-base catalysis and the reaction is endothermic. The active-site –CO2− groups of Glu-171 and Asp-297 (Glu-171–CO2− and Asp-297–CO2−) have been considered as the required general base catalysts in the bacterial o-quinoprotein methanol dehydrogenase (MDH) reaction. Based on quantum mechanics/molecular mechanics (QM/MM) calculations, the free energy for MeOH reduction of o-PQQ when MeOH is hydrogen bonded to Glu-171–CO2− and the crystal water (Wat1) is hydrogen bonded to Asp-297–CO2− is ΔG‡ = 11.7 kcal/mol, which is comparable with the experimental value of 8.5 kcal/mol. The calculated ΔG‡ when MeOH is hydrogen bonded to Asp-297–CO2− is >50 kcal/mol. The Asp-297–CO2−···Wat1 complex is very stable. Molecular dynamics (MD) simulations on MDH·PQQ·Wat1 complex in TIP3P water for 5 ns does not result in interchange of Asp-297–CO2− bound Wat1 for a solvent water. Starting with Wat1 removed and MeOH hydrogen bonded to Asp-297–CO2−, we find that MeOH returns to be hydrogen bonded to Glu-171–CO2− and Asp-297–CO2− coordinates to Ca2+ during 3 ns simulation. The Asp-297–CO2−···Wat1 of reactant complex does play a crucial role in catalysis. By QM/MM calculation ΔG‡ = 1.1 kcal/mol for Asp-297–CO2− general-base catalysis of Wat1 hydration of the immediate CH2O product → CH2(OH)2. By this means, the endothermic oxidation-reduction reaction is pulled such that the overall conversion of MeOH to CH2(OH)2 is exothermic.

Determination of enzyme mechanisms by molecular dynamics: Studies on quinoproteins, methanol dehydrogenase, and soluble glucose dehydrogenase

Molecular dynamics (MD) simulations have been carried out to study the enzymatic mechanisms of quinoproteins, methanol dehydrogenase (MDH), and soluble glucose dehydrogenase (sGDH). The mechanisms of reduction of the orthoquinone cofactor (PQQ) of MDH and sGDH involve concerted base‐catalyzed proton abstraction from the hydroxyl moiety of methanol or from the 1‐hydroxyl of glucose, and hydride equivalent transfer from the substrate to the quinone carbonyl carbon C5 of PQQ. The products of methanol and glucose oxidation are formaldehyde and glucolactone, respectively. The immediate product of PQQ reduction, PQQH− [−HC5(O−) −C4( = O) −] and PQQH [−HC5(OH) −C4( = O) −] converts to the hydroquinone PQQH2 [−C5(OH) = C4(OH) −]. The main focus is on MD structures of MDH • PQQ • methanol, MDH • PQQH−, MDH • PQQH, sGDH • PQQ • glucose, sGDH • PQQH− (glucolactone, and sGDH • PQQH. The reaction PQQ → PQQH− occurs with Glu 171–CO2− and His 144–Im as the base species in MDH and sGDH, respectively. The general‐base‐catalyzed hydroxyl proton abstraction from substrate concerted with hydride transfer to the C5 of PQQ is assisted by hydrogen‐bonding to the C5 = O by Wat1 and Arg 324 in MDH and by Wat89 and Arg 228 in sGDH. Asp 297–COOH would act as a proton donor for the reaction PQQH− → PQQH, if formed by transfer of the proton from Glu 171–COOH to Asp 297–CO2− in MDH. For PQQH → PQQH2, migration of H5 to the C4 oxygen may be assisted by a weak base like water (either by crystal water Wat97 or bulk solvent, hydrogen‐bonded to Glu 171–CO2− in MDH and by Wat89 in sGDH).

Conformations and dynamics of Ets-1 ETS domain–DNA complexes

Molecular dynamics studies have been performed for 3.5 ns on the ETS domain of Ets-1 transcription factor bound to the 14-bp DNA, d(AGTGCCGGAAATGT), comprising the core sequence of high-affinity (GGAA), ETS–GGAA. In like manner, molecular dynamics simulations have been carried out for 3.9 ns on the mutant low-affinity core sequence, GGAG (ETS–GGAG). Analyses of the DNA backbone of ETS–GGAG show conformational interconversions from BI to BII substates. Also, crank shaft motions are noticed at the mutated nucleotide base pair step after 1,500 ps of dynamics. The corresponding nucleotide of ETS–GGAA is characteristic of a BI conformation and no crank shaft motions are observed. The single mutation of ETS–GGAA to ETS–GGAG also results in variations of helical parameters and solvent-accessible surface area around the major and minor grooves of the DNA. The presence of water contacts during the entire simulation proximal to the fourth base pair step of core DNA sequence is a characteristic feature of ETS–GGAA. Such waters are more mobile in ETS–GGAG at 100 ps and distant after 1,500 ps. Anticorrelated motions between certain amino acids of Ets-1 protein are predominant in ETS–GGAA but less so or absent in the mutant. These motions are reflected in the flexibility of amino acid residues of the protein backbone. We consider that these conformational features and water contacts are involved in stabilizing the hydrogen bond interactions between helix-3 residues of Ets-1 and DNA during the transcription process.