(B-53) Molecular Dynamics Simulations of Glucose Oxidase Reveal Alternate Dimer Forms with Potential for Better Direct Electron Transfer to Electrode Surfaces

Introduction:: Glucose oxidase (GOx), a dimeric enzyme with FAD redox cofactors from Aspergillus niger, oxidizes D-glucose into gluconolactone and reduces O₂ to H₂O₂¹. GOx can be immobilized on an electrode, and the electrons in these two half reactions can be transferred to the electrode to generate a current. This is the foundation of GOx-based glucose sensors, which have important biomedical applications, such as monitoring diabetic patients’ blood glucose. Performance of GOx-based glucose biosensors is suboptimal because the active site of GOx is buried within the dimer interface, hindering direct electron transfer (DET) from the active site to the electrode. However, there is an alternate form of the GOx dimer, with a more exposed active site, which has greatly improved DET². The atomic structure of this alternate GOx dimer form is not known. In this study, we use molecular modelling and molecular dynamics simulations to investigate the following questions (1) What are the plausible alternate forms of GOx dimer? (2) Which alternate forms of the GOx dimer are comparable in stability to the native form? (3) Of the stable alternate forms of the GOx dimer, which is likely to have better DET? Information obtained in this study will be used to engineer GOx for improved DET.


Materials and Methods:: The crystal structure of the GOx dimer with FAD cofactors from A. niger [pdb #1CF3]¹ was used as a template. A plausible alternate dimer form was obtained using UCSF Chimera³. Amber general forcefield gaff2⁴ parameters for FAD were generated with the Antechamber⁵ and parmchk2 programs. These parameters were assigned to FAD and the Amber protein forcefield ff14SBonlysc⁶ parameters were assigned to the protein structure using the LEaP program. The native and the alternate dimer forms were subjected to simulations with the generalized Born solvent model⁷ using the Amber pmemd simulation program^8,9. The lifetime of intermolecular contacts between GOx monomers and the distance between the center of mass of GOx monomers were measured over the course of the simulations using the cpptraj program¹⁰.

Results, Conclusions, and Discussions:: During the simulation of the native GOx dimer, several intermolecular contacts persisted, and the distance between the center-of-mass of both monomers was maintained at an average of ~40.0 Å. During the simulations of an alternate form of the dimer, all the contacts were lost and the distance between center-of-mass of monomers grew well over 40 Å as the monomers dissociated. On several instances, the distance between center-of-mass of monomers shrank as the monomers reassociated in new conformations, forming new contacts. Among these new conformations there was a range of stability, some approaching the stability of the native form, and the active sites were more exposed than in the native form.

In this study, we demonstrated that the GOx dimer can exist in marginally stable alternate forms that have more exposed active sites. These alternate forms could adsorb to electrode surfaces in a way that promotes better DET to the electrode surface. To develop GOx-based glucose sensors with better DET and therefore better performance, efforts should be made to engineer GOx to favor alternate dimer forms over the native form.


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