Abstract

The nitro-explosive compounds 2,4,6-trinitrotoluene, 2,4,6-trinitrophenol, and 1,2,3-trinitroglycerol are persistent environmental contaminants. The presence of different functional groups in these molecules represents a great challenge to enzymatic catalysis. The chemical variety of these three substrates is such that they do not bind and interact with catalytic residues within an enzyme with the same affinity. In this context, two Xenobiotic Reductase enzymes produced by the bacteria Pseudomonas putida can catalyze the reduction of these compounds with different affinities and regioselectivity. The structural bases that support this substrate promiscuity and catalytic preferences are unknown. Therefore, through molecular dynamics simulations and free energy calculations, we explored the structural properties driving the specific interactions of these enzymes with their substrates and cofactor. Models of Xenobiotic Reductase A and B enzymes in complex with 2,4,6-trinitrotoluene, 2,4,6-trinitrophenol, or 1,2,3-trinitroglycerol were built, and the ligand enzyme interaction was simulated by molecular dynamics. The structural analysis of the molecular dynamics simulations shows that loops 3, 5, 7, 9, 11, and 13 of Xenobiotic Reductase B, and loops 4, 5, 7, 11, 13, and 15 Xenobiotic Reductase A, are in contact with the ligands during the first stages of the molecular recognition. These loops are the most flexible regions for both enzymes; however, Xenobiotic Reductase B presents a greater range of movement and a higher number of residues interacting with the ligands. Finally, the distance between the cofactor and the different reactive groups in the substrate reflects the regioselectivity of the enzymes, and the free energy calculations are consistent with the substrate specificity of both enzymes studied. The simulation shows a stable interaction between the aromatic ring of the substrates and Xenobiotic Reductase B. In contrast, a less stable interaction with the different nitro groups of the aromatic ligands was observed. In the case of 1,2,3-trinitroglycerol, Xenobiotic Reductase B interacts more closely with the nitro groups of carbon 1 or 3, while Xenobiotic Reductase A is more selective by nitro groups of carbon 2. The obtained results suggest that the flexibility of the loops in Xenobiotic Reductase B and the presence of polar and aromatic residues present in loops 5 and 7 are fundamental to determine the affinity of the enzyme with the different substrates, and they also contribute to the proper orientation of the ligands that directs the catalytic reaction.