Possible structures of the active site of a metalloglutathione transferase ลฝ . enzyme FosA are deduced from ab initio calculations. The active site of the manganese ลฝ . fosfomycin resistance protein FosA was reported, from electron paramagnetic resonance ลฝ . EPR studies, to have three waters bound in the first shell of the active site. Sequence analysis with two related enzymes, extradiol dioxygenase and glyoxalase I, identified two conserved ligand binding residues, a glutamate and a histidine, but the available experimental data do not identify the third protein ligand. A number of previous studies have found that a metalloenzyme active site is inherently determined by the in vacuo optimized structure of the first shell complex and only weakly perturbed by the surrounding protein environment. An ab initio optimized structure of the active site of extradiol dioxygenase is in good agreement with the X-ray structure and adds support to this observation. Starting with six ligands that include one glutamate and two histidines, four-, five-, and six-coordinated first-shell active-site structures are calculated to be energetically competitive with the four coordinate structures generally lower in energy.
If two glutamate and one histidine ligands are assumed to model FosA, then a sixcoordinated structure with three internally hydrogen bonded waters is calculated with manganese. Ab initio calculation finds that both the two glutamate and one histidine and the two histidine and one glutamate model are compatible with the binding of three waters in the first shell. The choice between the two models is dependent on the mode of binding and the number of waters displaced by the substrate. The binding of glutathione and the enzyme mechanism is discussed based on the theoretically predicted sites.