Purification, substrate range, and metal center of AtzC: the N-isopropylammelide aminohydrolase involved in bacterial atrazine metabolism.
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 micro M 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 (Delta epsilon = 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.
Shapir
Nir
N
Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, St. Paul, Minnesota 55108, USA.
Osborne
Jeffrey P
JP
Johnson
Gilbert
G
Sadowsky
Michael J
MJ
Wackett
Lawrence P
LP
D000581:Amidohydrolases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D000595:Amino Acid Sequence; D000619:Aminohydrolases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D001280:Atrazine / Q000378:metabolism*; D001426:Bacterial Proteins; D001665:Binding Sites; D004578:Electron Spin Resonance Spectroscopy; D007700:Kinetics; D008969:Molecular Sequence Data; D011549:Pseudomonas / Q000201:enzymology* / Q000235:genetics; D016415:Sequence Alignment; D013057:Spectrum Analysis / Q000379:methods; D013379:Substrate Specificity; D015032:Zinc / Q000737:chemistry
D001426:Bacterial Proteins; D000581:Amidohydrolases; D000619:Aminohydrolases; C109894:AtzC protein, Pseudomonas; D015032:Zinc; D001280:Atrazine