Deamination is ubiquitous in nature and has important biological significance. Leucobacter triazinivorans JW-1, recently isolated from sludge, can rapidly degrade s-triazine herbicides. The responsible enzymes, however, have not been purified and characterized.Herein, we purified an amidohydrolase, i.e., N-isopropylammelide isopropylaminohydrolase (AtzC) from JW-1 cells by ammonium sulfate precipitation and three chromatography steps. The purified AtzC catalyzed amidohydrolysis of N-isopropylammelide to cyanuric acid. The optimal catalytic conditions of the purified AtzC were 42 °C and pH 7.0, and the Kₘ and Vₘₐₓ of AtzC was 0.811 mM and 28.19 mmol/min·mg. AtzC could catalyze amidohydrolysis of an N-alkyl substituent from dihydroxy s-triazines to cyanuric acid. Molecular docking and structural alignments were used to infer AtzC catalytic mechanism. The structural architecture of AtzC resembled that of cytosine deaminase in class III amidohydrolase, with a single Zn²⁺ coordinated by His and Asp. Interestingly, the AtzC lacks an acidic residue putatively to activate water for hydrolysis as compared to the other amidohydrolases. His253 in AtzC probably functions as a single general acid-base catalyst. These findings further enhance our understanding how aminohydrolases catalyze the metabolism of s-triazine herbicides.