Purification, substrate range, and metal center of AtzC: The N-isopropylammelide aminohydrolase involved in bacterial atrazine metabolism

Nir Shapir, Jeffrey P. Osborne, Gilbert Johnson, Michael J. Sadowsky, Lawrence P. Wackett

Research output: Contribution to journalArticlepeer-review

42 Scopus citations

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 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 (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.

Original languageEnglish (US)
Pages (from-to)5376-5384
Number of pages9
JournalJournal of bacteriology
Volume184
Issue number19
DOIs
StatePublished - Oct 2002

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Copyright:
Copyright 2008 Elsevier B.V., All rights reserved.

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