Abstract
Extradiol dioxygenases facilitate microbial aerobic degradation of catechol and its derivatives by activating molecular dioxygen and incorporating both oxygen atoms into their substrates. Experimental and theoretical studies have focused on the mechanism of the reaction at the active site. However, whether the catalytic rate is limited by O2 access to the active site has not yet been explored. Here, we choose a recently solved X-ray structure of homoprotocatechuate 2,3-dioxygenase as a typical example to determine potential pathways for O2 migration from the solvent into the enzyme center. On the basis of the trajectories of two 10-ns molecular dynamics simulations, implicit ligand sampling was used to calculate the 3D free energy map for O2 inside the protein. The energetically optimal routes for O2 diffusion were identified for each subunit of the homotetrameric protein structure. The O2 tunnels formed because of thermal fluctuations were also characterized by connecting elongated cavities inside the protein. By superimposing the favorable O2 tunnels on to the free energy map, both energetically and geometrically preferred O2 pathways were determined, as also were the amino acids that may be critical for O2 passage along these paths. Our results demonstrate that identical subunits possess quite distinct O2 tunnels. The order of O2 affinity of these tunnels is generally consistent with the order of the catalytic rate of each subunit. As a consequence, the probability of finding the reaction product is highest in the subunit containing the highest O2 affinity pathway.
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This work was supported by the Youth Foundation of DLUT (893103), National Natural Science Foundation (10772042), and the National Basic Research Program (2009CB918501) of China.
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Xu, L., Zhao, W. & Wang, X. Finding molecular dioxygen tunnels in homoprotocatechuate 2,3-dioxygenase: implications for different reactivity of identical subunits. Eur Biophys J 39, 327–336 (2010). https://doi.org/10.1007/s00249-009-0551-9
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DOI: https://doi.org/10.1007/s00249-009-0551-9