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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

Nir Shapir,1,2,3 Jeffrey P. Osborne,1,2 Gilbert Johnson,1 Michael J. Sadowsky,2,3,4 and Lawrence P. Wackett1,2,4*

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 Km and kcat 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 kcat 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 ({Delta}{varepsilon} = 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.

* Corresponding author. Mailing address: Department of Biochemistry, Molecular Biology and Biophysics, 140 Gortner Lab, 1479 Gortner Ave., University of Minnesota, St. Paul, MN 55108. Phone: (612) 625-3785. Fax: (612) 625-5780. E-mail: wackett{at}biosci.cbs.umn.edu.

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.



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