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Journal of Bacteriology, October 2002, p. 5376-5384, Vol. 184, No. 19
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.19.5376-5384.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

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

Department of Biochemistry, Molecular Biology and Biophysics,¹ BioTechnology Institute,² Center for Microbial and Plant Genomics,⁴ Department of Soil, Water & Climate, University of Minnesota, St. Paul, Minnesota 55108³

Received 12 April 2002/ Accepted 10 July 2002

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 ( = 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.

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