Theoretical Studies On Molecular Mechanism For Oxygen Diffusion And Activation In 1-H-3-hydroxy-4-oxoquinaldine-2,4-dioxygenase(HOD)

1-H-3-hydroxy-4-oxoquinaldine-2,4-dioxygenase(HOD)is one of specific dioxygenases that do not require any cofactors to bind and activate oxygen.Recently it has been reported that oxygen molecule can enter active center along a specific channel of certain oxidases and oxygenases,rather than conventional free diffusion.This work studied the oxygen diffusion and activation processes of HOD to understand the detailed mechanism for its unique degradation of the heterocyclic aromatic substrate.The first part of the thesis discussed the catalytic molecular mechanism for the HOD-catalyzed reaction.HOD catalyzes the oxidation of 1-H-3-hydroxyl-4-oxo-quinaldine to form N-acetylanthranilate acid and carbon monoxide,which is a key step in the degradation of quinaldine.The potential energy surface of the catalytic process was carefully calculated by a quantum mechanics method,which revealed that the activation of oxygen was the rate-limiting step in the catalytic oxygenation.Furthermore,frontier orbital analysis revealed that the oxygen molecule was activated to a singlet peroxide intermediate and form C-O bonds with the substrate in the catalytic packet.First,1-H-3-hydroxyl-4-oxo-quinaldine deprotoned with assistance of His251 and Asp126,and then electrons of substrate anion gradually transferred to dioxygen,so that dioxygen was able to conduct a spin flip from triplet to singlet state.The effects of the oxygen binding mode,the active sites of Trp36 and His102,and the chemical properties of the substrate on oxygen activation were investigated.It was found that all of these factors casted certain influences on the oxygen activation,suggesting that the microscopic structure should regulate the non-cofactor oxygen activation in HOD.The second part discussed the oxygen diffusion pathways in HOD.25 potential oxygen channels were predicted by the Caver software,and five dominant channels were selected as the main research objects.Then MDpocket software found some pockets on the surface of HOD.According to previous studies,most of oxygen channels can pass active pockets,so one of the most possible channels in HOD was proposed.The channel located on Re surface of the heterocyclic aromatic substrate,and the oxygen binding site was under the Re surface's oxygen hole consisting of skeleton of the Trp36 and His102.The oxygen diffusion pathway was further studied with the random acceleration molecular dynamics simulation,giving a consistent result with the channel analysis.The umbrella sampling calculation confirmed that dioxygen would access the active site via the channel feasibly,based on the free energy scanning.The oxygen diffusion analysis provided an in-depth understanding on the regio-and stereo-selectivity of HOD.Overall,the catalytic mechanism of HOD was studied by theoretical calculation,and the unique oxygen diffusion pathway was proposed as well.In particular,the oxygen activation of HOD was illustrated at the level of electronic structure with the frontier orbital analysis.The Caver and MDpocket prediction,random accelerated molecular dynamics simulation,and umbrella sampling dynamics simulation method were integrated to unveil the most possible oxygen channels.These detailed understandings on the oxygen diffusion and activation would be helpful for future application in bio-degradation of aromatic compounds.