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Journal of Bacteriology, January 2003, p. 126-134, Vol. 185, No. 1
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.1.126-134.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Metal Ion Dependence of Recombinant Escherichia coli Allantoinase

Departments of Microbiology & Molecular Genetics and Biochemistry & Molecular Biology, Michigan State University, East Lansing, Michigan 48824-4320

Received 29 July 2002/ Accepted 1 October 2002

Allantoinase is a suspected dinuclear metalloenzyme that catalyzes the hydrolytic cleavage of the five-member ring of allantoin (5-ureidohydantoin) to form allantoic acid. Recombinant Escherichia coli allantoinase purified from overproducing cultures amended with 2.5 mM zinc, 1 mM cobalt, or 1 mM nickel ions was found to possess 1.4 Zn, 0.0 Co, 0.0 Ni, and 0.4 Fe; 0.1 Zn, 1.0 Co, 0.0 Ni, and 0.2 Fe; and 0.0 Zn, 0.0 Co, 0.6 Ni, and 0.1 Fe per subunit, respectively, whereas protein obtained from nonamended cultures contains near stoichiometric levels of iron. We conclude that allantoinase is incompletely activated in the recombinant cells, perhaps due to an insufficiency of a needed accessory protein. Enzyme isolated from nonsupplemented cultures possesses very low activity (k_cat = 34.7 min^-1) compared to the zinc-, cobalt-, and nickel-containing forms of allantoinase (k_cat values of 5,000 and 28,200 min^-1 and 200 min^-1, respectively). These rates and corresponding K_m values (17.0, 19.5, and 80 mM, respectively) are significantly greater than those that have been reported previously. Absorbance spectroscopy of the cobalt species reveals a band centered at 570 nm consistent with five-coordinate geometry. Dithiothreitol is a competitive inhibitor of the enzyme, with significant K_i differences for the zinc and cobalt species (237 and 795 µM, respectively). Circular dichroism spectroscopy revealed that the zinc enzyme utilizes only the S isomer of allantoin, whereas the cobalt allantoinase prefers the S isomer, but also hydrolyzes the R isomer at about 1/10 the rate. This is the first report for metal content of allantoinase from any source.

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