Metal ion dependence of recombinant Escherichia coli allantoinase

Abstract

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 approximately 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 micro M, 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.

FIG. 1.

Allantoinase reaction in the context of soybean uric acid metabolism. The boxed reaction is catalyzed by allantoinase.

FIG. 2.

Sequence comparison of regions of selected amidohydrolases. Regions of sequence similarity are shown for allantoinases (All) of Escherichia coli, Deinococcus radiodurans (D.rad.), Bacillus subtilis (B.sub.), Bacillus halodurans (B.hal.), Streptomyces coelicolor (S.coe), and Saccharomyces cerevisiae (S.cer.); hydantoinases (Hyd) of Agrobacterium tumefaciens (A.tum.), Ralstonia pickettii (R.pic.), Agrobacterium sp. strain IP I-671 (A. sp.), Geobacillus stearothermophilus (formerly Bacillus stearothermophilus [G.ste.]), S. coelicolor, Brucella melitensis (B.mel.), Pseudomonas putida (P.put.), Sulfolobus tokodaii (S.tok.), a Thermus sp. (Ther.), and Arthrobacter aurescens (A.aur.); E. coli dihydroorotase (DHO), and Klebsiella aerogenes urease large subunit (K.aer. Ure). Crystal structures have been reported for the D-specific hydantoinases from Thermus (2) and G. stearothermophilus (8) (Protein DataBank accession no. 1GKP and 1K1D), the L-specific hydantoinase from A. aurescens (1) (accession no. 1GKR), E. coli dihydroorotase (47) (accession no. 1J79), and K. aerogenes urease (20) (accession no. 1FJW). The metallocenter ligands in these proteins are underlined with carbamylated lysines bridging the two metals.

FIG. 3.

Absorbance spectra of allantoinases prepared from cobalt (solid line), or zinc (dashed-dotted line)-amended cultures. (Inset) Expanded absorbance scale of the 400- to 750-nm region. The concentration of enzymes were 20 μM for the zinc form and 15 μM for the cobalt form in 50 mM HEPES (pH 7.8) buffer. The concentrations of enzymes in the inset were 400 μM for the zinc form and 300 μM for the cobalt form.

FIG. 4.

Effects of pH on allantoinase stability and activity. Open symbols represent results for the cobalt form of the enzyme (corresponding to the right axis), and solid symbols represent those for the zinc form (corresponding to the left axis). The buffers used were MES (circles), HEPES (squares), CHES (triangles), and CAPS (inverted triangles). (A) Stability of allantoinases for 1 h at room temperature as a function of pH. (B) Activity as a function of pH. The curves are fit to the data in the range of pH 7.3 to 10.5 yielding pK_a values of 9.0 for the cobalt form of allantoinase (dashed line) and 8.8 for the zinc form (solid line).

FIG. 5.

DTT inhibition of allantoinase. (A) Zinc form of allantoinase. Allantoin concentrations were 50 mM (•), 30 mM (▪), 15 mM (▴), and 7 mM (▾). (B) Cobalt form of allantoinase. The allantoin concentrations were 80 mM (•), 50 mM (▪), 25 mM (▴), and 10 mM (▾).

FIG. 6.

Allantoinase assays monitored by CD spectropolarimetry. (A) Allantoinase prepared from zinc-amended cultures. (B) Allantoinase from cobalt-amended cultures.