TrzN from Arthrobacter aurescens TC1 Is a Zinc Amidohydrolase

Chemicals.All buffers and routine biochemicals were obtained from Sigma-Aldrich and were of the highest purity available. The following compounds were graciously provided by Syngenta (Greensboro, N.C.): atrazine, simazine, terbutylazine, and ametryn. Simetryn was obtained from Sigma-Aldrich. All other compounds were synthesized in our laboratory. Cyanoatrazine was synthesized as previously described (15), and thioatrazine was prepared by refluxing atrazine, thiourea, and water in ethanol under a nitrogen atmosphere as previously described (20).

Expression and purification of TrzN.Plasmid pAG, containing the chaperones groEL and groES (6), was transformed into E. coli BRL21(DE3) containing plasmid pET28b+::trzN (20). Strain BRL21(DE3)(pET28b+::trzN) (pAG) was grown in LB medium (13) containing kanamycin (50 μg/ml) and chloramphenicol (30 μg/ml) at 15°C, with shaking at 150 rpm. When cultures reached an optical density of 0.5 at 600 nm, the chaperones were induced by the addition of 0.0015% (wt/vol) l-arabinose, and 1.5 μM IPTG (isopropyl-β-d-thiogalactopyranoside) was added after an additional 90 min of incubation at 15°C. Induced cells were grown for an additional 16 h under the same conditions, cultures were centrifuged at 10,000 × g for 10 min at 4°C and washed three times with 0.85% (wt/vol) NaCl, and cell pellets were resuspended in 30 ml of 0.1 M sodium phosphate buffer, pH 7.0, containing 10% glycerol. Cells were broken by passage, three times, through a chilled French pressure cell operated at 140 MPa, and cell extracts were obtained by centrifugation at 18,000 × g for 90 min at 4°C. Lysates were applied to a 5-ml HisTrap chelating column (Amersham Pharmacia Biotech, Piscataway, NJ), complexed with Ni²⁺, according to the manufacturer's instructions. The column was washed with 15 ml 0.1 M sodium phosphate buffer, pH 7.0, followed by two washes with the same buffer supplemented with 0.1 M and 0.25 M imidazole, respectively. All buffers contained 10% glycerol. Enzyme was eluted from the column with 15 ml of 0.5 M imidazole in 0.1 M sodium phosphate buffer, pH 7.0, and the purified enzyme was concentrated using a Centricon-30 filtration unit (Amicon, Beverly, MA). Imidazole was removed from the enzyme preparation by dialysis, twice, at 4°C for 4 h against 4 liters of 0.1 M sodium phosphate buffer, pH 7.0, containing 10% glycerol. Enzyme purity and subunit molecular weight were estimated by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (13).

Enzyme assay.Enzyme activity was measured by monitoring the disappearance of the substrate ametryn at 264 nm by using a Beckman DU 640 spectrophotometer (Beckman Coulter, Fullerton, CA). The product, hydroxyatrazine, had negligible absorbance at this wavelength. Reactions (1 ml) were carried out at 37°C in 0.1 M sodium phosphate buffer, pH 8.0, containing 132 μM ametryn. Reactions were initiated by the addition of enzyme, and the molar absorbance at 264 nm for ametryn under these conditions was determined to be 5 mM⁻¹ cm⁻¹. Enzyme activity against other s-triazines was determined using the same procedure, with the following changes in wavelengths and molar absorbance (ε) values used for measuring and calculating substrate disappearance: atrazine, λ = 264 nm, ε = 3.5 mM⁻¹ cm⁻¹; dipropetryn, λ = 255 nm, ε = 7.5 mM⁻¹ cm⁻¹; simetryn, λ = 264 nm, ε = 5.1 mM⁻¹ cm⁻¹; terbutylazine, λ = 264 nm, ε = 2.7 mM⁻¹ cm⁻¹; simazine, λ = 264 nm, ε = 3.1 mM⁻¹ cm⁻¹; and cyanoatrazine, λ = 300 nm, ε = 2.1 mM⁻¹ cm⁻¹.

Gel filtration chromatography.The holoenzyme molecular weight was estimated by gel filtration chromatography on a Superose 12 HR column using a fast protein liquid chromatograph system (Pharmacia, Uppsala, Sweden). The column was equilibrated with 0.1 M sodium phosphate buffer, pH 7.0, containing 0.15 M KCl, at a flow rate of 0.3 ml min⁻¹. The column was calibrated with proteins and compounds of known molecular weight: thyroglobulin, 670,000; gamma globulin, 158,000; chicken ovalbumin, 44,000; horse myoglobin, 17,000; and vitamin B₁₂, 1,350.

Influence of growth medium Zn²⁺ concentration on TrzN activity.The effect of Zn²⁺ concentration in the growth medium on TrzN activity and enzyme metal content was evaluated by supplementing LB medium with 0, 20, 100, or 500 μM ZnSO₄. Cells were grown and the enzyme purified as described above. The specific activity of TrzN, using ametryn as substrate, was calculated as described above, and the metal concentration in enzyme preparations was determined by using inductively coupled plasma emission spectroscopy. For metal analyses, purified TrzN (2.5 mg) was dissolved in 5 ml 0.1 M sodium phosphate buffer acidified with 10% (wt/vol) HCl. Samples were incubated overnight at 90°C, and metal content was determined by inductively coupled plasma emission spectroscopy analysis at the University of Minnesota Soils Analytical Laboratory (St. Paul, MN).

Effect of chelators on TrzN activity.Purified TrzN (25 μM) was incubated at 25°C with 5 mM of 1,10-phenanthroline, 8-hydroxyquinoline-5-sulfonic acid, or EDTA, and TrzN activity was measured with ametryn as substrate, over time, as described above.

Metal-depleted enzyme and metal reconstitution.Metal-depleted TrzN was prepared by incubating purified enzyme with 5 mM 8-hydroxyquinoline-5-sulfonic acid for 8 h at 25°C. Protein was separated from free or metal-bound 8-hydroxyquinoline-5-sulfonic acid by passage through a Sephadex G-25 column (1.5 by 30 cm) equilibrated with 0.1 M sodium phosphate buffer, pH 7.0. Metal-depleted enzyme eluted from the column was concentrated using a Centriprep YM-30 filtration unit (Amicon, Beverly, MA). Holoenzyme (25 μM) and metal-depleted enzyme (70 μM) were incubated in 0.1 M sodium phosphate buffer, pH 7.0, containing 0.2 mM ZnSO₄, CuCl₂, FeSO₄, MnSO₄, CoCl₂, or NiCl₂ for 30 min at 25°C. Enzyme activity was measured as described above using ametryn as substrate. Reaction mixtures were supplemented with 1.5 mM dithiothreitol to maintain iron as Fe(II). Enzyme activity was reported as the percentage of the initial activity.

The Co(II)- and Zn(II)-reconstituted TrzN protein was prepared by incubating 35 μM of metal-depleted enzyme with 250 μM of CoCl₂ or ZnSO₂. At selected time points, the activities of the reconstituted enzymes were measured using ametryn. In addition, the specific activity of the enzymes, following 2 h of reconstitution with metals, was determined using ametryn, atrazine, dipropetryn, and cyanoatrazine as substrates as described above.

Electronic spectra of Co(II)-reconstituted enzyme.Approximately 400 μM of metal-depleted TrzN was incubated for 30 min at 25°C with 300 μM CoCl₂ (0.75 equivalents per TrzN subunit). Visible spectra (300 to 800 nm) of samples were acquired using a Beckman DU 640 spectrophotometer (Beckman Instruments, Fullerton, CA). The calculated extinction coefficient for Co(II)-reconstituted enzyme was based on moles of enzyme subunit. Controls contained metal-depleted enzyme alone, buffer alone, and buffer plus CoCl₂. The influence of the competitive inhibitor thioatrazine on metal-enzyme spectra was determined as described above with 30 μM thioatrazine added.

Kinetic parameters for selected s-triazine herbicides.Solutions of different s-triazine herbicides were prepared in 0.1 M sodium phosphate buffer, pH 8.0. The compounds were incubated with purified TrzN, and changes in absorbance were recorded. The wavelengths used were as described above. Kinetic parameters were calculated from initial hydrolysis rates at different concentrations of substrate. Control samples without enzyme were analyzed in parallel, and no spontaneous activity was detected. Kinetic parameters of TrzN were calculated using the Hanes-Woolf equation: [S]/V₀ = [S]/V_max + K_m/V_max (17). Linear regression of the plot [S]/V₀ versus [S] was used to determine the V_max and K_m parameters, and k_cat was calculated by dividing V_max by the moles of TrzN subunits present. For steady-state kinetic determinations, substrate concentrations ranged from 1.3 μM to 140 μM.

Sequence comparison of TrzN with amidohydrolase superfamily proteins.Modest sequence identity was observed in comparisons of TrzN (accession no. AAS20185) with AtzA (accession no. NP_862474), AtzB (accession no. P95442), AtzC (accession no. O52063), and E. coli cytosine deaminase (CodA) (accession no. P25524) (Fig. 2). The Atz proteins have been established to be members of the amidohydrolase superfamily (11, 16). This superfamily contains a number of metalloenzymes with known X-ray structures (18), of which cytosine deaminase is the most closely related to the atrazine enzymes in sequence alignments. The alignments suggest the presence of three conserved histidine residues known to provide ligands to a metal in cytosine deaminase (4). The remaining metal ligand(s) is more ambiguous. However, the presence of at least three ligands and the overall sequence relatedness suggested that TrzN may be a metalloenzyme. To investigate this more fully and to determine the identity of the putative metal and its stoichiometry, significant quantities of high-activity TrzN were needed. This, in turn, required the use of a highly efficient protein expression system, and that is described below.

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FIG. 2.

Multiple sequence alignment of TrzN, AtzB, AtzA, AtzC, and CodA showing residues near known histidine ligands to the mononuclear metal center of E. coli cytosine deaminase (CodA). The numbers at the right signify the percent sequence identity of each protein in a pairwise alignment with TrzN.

TrzN expression, purification, and general characterization.TrzN activity was low in initial clones containing plasmid pET28b+::trzN (20). Subsequent transformation of plasmid pAG (6) expressing the chaperone proteins GroEL and GroES into E. coli (pET28b+::trzN) yielded cells expressing significant levels of ametryn degradation activity in crude lysates. A major protein band was observed by SDS-PAGE that migrated slightly faster than the intense bands of the chaperone proteins (data not shown). Ultimately, conditions were found to elute the His-tagged TrzN uniquely from a nickel column to yield a homogeneous protein. The enzyme was dialyzed to remove imidazole, which inhibited TrzN activity. Ten milligrams of pure protein was obtained from 1 liter culture. Yield was sacrificed to obtain purity.

The purified TrzN protein appeared homogeneous by SDS-PAGE, with an estimated subunit molecular weight of 53,000. Based on translation of the gene sequence, the native TrzN protein with the His tag was calculated to have a subunit molecular weight of 52,991. Gel filtration indicated a holoenzyme molecular weight of 50,000, suggesting that the native protein was a monomer. These data reveal that, if TrzN is a metalloenzyme, all the ligands to the metal(s) are provided by amino acids in a single polypeptide chain.

Metal determination experiments with native enzyme.In initial experiments, substoichiometric metal concentrations were detected, with zinc being observed in the greatest abundance. Addition of ZnCl₂ or CoCl₂ resulted in substantial increases in activity. The largest activity increases were observed with zinc, which, at saturation, typically yielded a specific activity of around 20 μmol/min per mg protein. However, different batches of purified, native TrzN showed highly variable specific activity, ranging from 1 to 20 μmol/min per mg protein.

In an attempt to overcome this variability, batches of recombinant E. coli cells were grown with increasing amounts of ZnSO₄ in the medium and TrzN was purified from each of the harvested cell pastes. The purified enzymes were then compared with respect to specific activity and zinc content (Table 1). A dramatic and asymptotic increase in specific activity of TrzN was observed with increasing zinc concentration in the growth medium. The highest specific activity of TrzN, obtained from cells grown with 500 μM zinc, was 22.2 μmol/min per mg protein, which is comparable to the maximum activity observed in the previously described zinc supplementation experiments. Metal determinations showed that the increase in specific activity was matched by a corresponding increase in the concentration of zinc in TrzN. The maximum activity was observed with a stoichiometry of one atom of zinc per enzyme subunit molecule.

Metal chelators remove metal and activity concomitantly.Native TrzN with high specific activity was treated with chelators or incubated without chelator under identical conditions as a control. Minimal diminution in activity was observed in the control or EDTA treatments. However, treatment with 1,10-phenanthroline or 8-hydroxyquinoline-5-sulfonic acid led to significant losses in enzyme activity over an 8-h period (Fig. 3). In subsequent experiments, TrzN was incubated with 8-hydroxyquinoline-5-sulfonic acid for 6 h, and activity and metal content were determined. After treatment, activity was approximately 4% of the original activity, and the zinc stoichiometry was less than 0.07 Zn per enzyme subunit. This preparation is referred to as “metal-depleted TrzN” in subsequent experiments.

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FIG. 3.

Time-dependent loss of TrzN ametryn-hydrolysis activity following incubation with 5 mM metal chelators or a buffer control. Symbols: ⧫, EDTA; ▴, 1,10-phenanthroline; ▪, 8-hydroxyquinoline-5-sulfonic acid; •, buffer control.

Metal addition to native and metal-depleted TrzN.The effect of zinc, cobalt, copper, nickel, manganese, and iron addition on enzyme activity was tested with native (Fig. 4A) and metal-depleted (Fig. 4B) TrzN. Very little effect on the native enzyme was seen with any metal, except for copper, which showed marked inhibition (Fig. 4A). Metal addition to metal-depleted TrzN showed three differential effects (Fig. 4B). Marked inhibition was observed with copper, even to levels well below that of uninhibited metal-depleted TrzN. Cobalt and zinc strongly stimulated activity, with zinc showing the greatest stimulation. In contrast, iron, manganese, and nickel showed little or no effect. The activity of the zinc-amended metal-depleted enzyme was within 90% of the activity of the original enzyme prior to metal depletion.

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FIG. 4.

Effect of metal addition on ametryn hydrolysis activity of native TrzN purified from recombinant cells grown on 500 μM zinc (A) and metal-depleted TrzN (B). All metals were added at a final concentration of 0.2 mM. Error bars represent the standard errors of the means.

Effect of metal on activity with different substrates.Native zinc-containing TrzN and cobalt-substituted TrzN were tested with several known substrates and one previously untested substrate (dipropetryn) (Fig. 5). Initially, metal-depleted enzymes were incubated with metals and tested for activity with different substrates over a period of time. As shown in the inset to Fig. 5, activity stabilized after about 30 min. Subsequent preincubations with metals were carried out for 2 h prior to assay with substrates. With ametryn and atrazine, cobalt-substituted enzyme contained 80% and 45%, respectively, of the activity of the native enzyme with zinc, which was set at 100%. With dipropetryn, the activity of the cobalt-substituted TrzN was only 30% of that of the native enzyme. With cyanoatrazine, activity was modestly higher with the cobalt-substituted enzyme than with the zinc-substituted enzyme.

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FIG. 5.

Specific activity of Zn(II)- (▨) and Co(II)-reconstituted TrzN (▦) with selected s-triazines as substrates. Inset: specific activity of metal-depleted TrzN with ametryn following incubation with cobalt or zinc for up to 6 h; solid circles indicate Co-TrzN, and solid squares represent Zn-TrzN. Error bars represent the standard errors of the means.

Steady-state kinetic parameters.In light of the experiments above, steady-state kinetic parameter assays were conducted with native enzyme that had one equivalent of zinc per subunit and negligible quantities of other metals. The substrates tested were major herbicides with which the activity is highest with zinc-containing TrzN (Table 2). The three lowest-activity substrates underwent chloride displacement. The k_cat and k_cat/K_m increased in the chlorinated s-triazine series: terbutylazine < simazine < atrazine. The higher-activity substrates contained an S-alkyl group rather than a chlorine atom. In general, the activities were approximately 1 order of magnitude higher for S-alkyl than for chloride displacement. The highest k_cat/K_m was observed with simetryn.

Thioatrazine (SH in place of the Cl atom of atrazine) was observed not to be a substrate for TrzN, but it did act as an inhibitor in admixture with atrazine or ametryn. Thioatrazine significantly inhibited TrzN activity; with both thioatrazine and ametryn present at 0.015 mM, activity was inhibited by 60%. Since the solubility is very limited (0.03 mM), extensive steady-state assays at several concentrations of thioatrazine as an inhibitor could not be conducted. However, using saturating thioatrazine concentrations and varying the ametryn concentration with and without inhibitor, two lines that intersected on the y axis were obtained on a double-reciprocal plot, suggesting that thioatrazine is a competitive inhibitor.

Electronic spectroscopy of cobalt-substituted TrzN.The electronic absorbance spectrum of 400 μM cobalt-substituted TrzN, prepared as described in Materials and Methods section, was obtained in buffered solution at pH 7.0 (Fig. 6). A comparison with metal-depleted enzyme allowed the calculation of a difference spectrum and an estimation of the extinction coefficient for the main absorption band of the cobalt center (Fig. 6, inset). The major band showed an absorption maximum at 550 nm with shoulders at 470, 520, and 600 nm. The extinction coefficient of the main band at 550 nm was 110 M⁻¹ cm⁻¹.

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FIG. 6.

UV-visible spectra of Co(II)-reconstituted (solid line) and metal-depleted (dotted line) TrzN. Inset: difference spectrum of Co(II)-reconstituted TrzN minus the spectrum of the metal-depleted enzyme.

Additional incubations were conducted with thioatrazine. The addition of thioatrazine increased the overall absorbance by 20%. Incubations of thioatrazine with cobalt salts alone or with metal-depleted enzyme did not give significant changes in absorbance.