Purification, Substrate Range, and Metal Center of AtzC: the N -Isopropylammelide Aminohydrolase Involved in Bacterial Atrazine Metabolism

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Abstract
N -Isopropylammelide isopropylaminohydrolase, AtzC, the third enzyme in the atrazine degradation pathway in Pseudomonas sp. strain ADP, catalyzes the stoichiometric hydrolysis of N -isopropylammelide to cyanuric acid and isopropylamine. The atzC gene was cloned downstream of the tac promoter and expressed in Escherichia coli , where the expressed enzyme comprised 36% of the soluble protein. AtzC was purified to homogeneity by ammonium sulfate precipitation and phenyl column chromatography. It has a subunit size of 44,938 kDa and a holoenzyme molecular weight of 174,000. The K m and k cat values for AtzC with N -isopropylammelide were 406 μM and 13.3 s −1 , respectively. AtzC hydrolyzed other N -substituted amino dihydroxy- s -triazines, and those with linear N -alkyl groups had higher k cat values than those with branched alkyl groups. Native AtzC contained 0.50 eq of Zn per subunit. The activity of metal-depleted AtzC was restored with Zn(II), Fe(II), Mn(II), Co(II), and Ni(II) salts. Cobalt-substituted AtzC had a visible absorbance band at 540 nm (Δε = 84 M −1 cm −1 ) and exhibited an axial electron paramagnetic resonance (EPR) signal with the following effective values: g ( x ) = 5.18, g ( y ) = 3.93, and g ( z ) = 2.24. Incubating cobalt-AtzC with the competitive inhibitor 5-azacytosine altered the effective EPR signal values to g ( x ) = 5.11, g ( y ) = 4.02, and g ( z ) = 2.25 and increased the microwave power at half saturation at 10 K from 31 to 103 mW. Under the growth conditions examined, our data suggest that AtzC has a catalytically essential, five-coordinate Zn(II) metal center in the active site and specifically catalyzes the hydrolysis of intermediates generated during the metabolism of s -triazine herbicides.