Metal Ion Dependence of Recombinant Escherichia coli Allantoinase
AUTOR(ES)
Mulrooney, Scott B.
FONTE
American Society for Microbiology
RESUMO
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 (kcat = 34.7 min−1) compared to the zinc-, cobalt-, and nickel-containing forms of allantoinase (kcat values of 5,000 and 28,200 min−1 and 200 min−1, respectively). These rates and corresponding Km 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 Ki 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.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=141845Documentos Relacionados
- Metal ion content of Escherichia coli versus cell age.
- Metal ion dependence of a heat-modifiable protein from the outer membrane of Escherichia coli upon sodium dodecyl sulfate-gel electrophoresis.
- Structural basis for the 3'-5' exonuclease activity of Escherichia coli DNA polymerase I: a two metal ion mechanism.
- Genetic Analysis of Streptomycin Dependence in ESCHERICHIA COLI
- Functional Expression and Characterization of the Two Cyclic Amidohydrolase Enzymes, Allantoinase and a Novel Phenylhydantoinase, from Escherichia coli