The 3D structure of the AmiF formamidase of Helicobacter pylori (denoted as AmiF(fhp)) is built by homology modeling. The docking studies show that AmiF(fhp) has restricted substrate specificity, as it only hydrolyzes formamide. In order to reveal the reaction mechanism and the catalytic role for Cys166, Lys133 and Asp168 in AmiF(fhp), three quantum mechanics' models are constructed based on the 3D structure of AmiF(fhp) and the reaction paths are obtained at B3LYP 6-31 + G* level. The calculated results show that (1) the reaction of Cysl66-formamide anion in the enzyme active proceeds via a transition state without the intervention of tetrahedral intermediate; (2) the positive charge on Lys133 polarizes the formamide in the TS region to redistribute the electron and thence decreases the free energy barrier; (3) the active site residue of Asp168 increases the free energy barrier as the negative charge will affect the electron distribution. (C) 2007 Elsevier Ltd. All rights reserved.