2.1 Materials
The resistance of water used was more than 18 M-ohms, which was purified by reverse osmosis followed by passage through a Millipore Reagent Water System (Millipore, Bedford, MA, USA). Octyl Sepharose CL-4B, chelating Sephacel (fast flow), and HiTrap Q columns were purchased from Amersham Biosciences (Taiwan Branch). Bis–Tris propane, PMSF, 5-hydantoinacetic acid, parabanic acid, phthalimide and 3-iminoisoindoline were purchased from Sigma (U.S.A.). EDTA, Tris–HCl, potassium phosphate, sodium chloride, sodium hydroxide, and zinc acetate were obtained from J.T. Baker (U.S.A.). Hydroxyapatite Sephacel was purchased from Biosepra (U.S.A.). Trypsin was purchased from Promega (U.S.A.). All other chemicals were obtained commercially at the highest purity possible.
2.2 Enzyme Standard Assay
A rapid spectrophotometric assay was used as the standard assay. Briefly, the decrease in absorbancy at 298 nm was measured upon hydrolysis of phthalimide as the substrate at 25℃.
To start the reaction, the enzyme solution was added into a 1 ml solution, containing 1 mM phthalimide and 100 mM Bis-Tris propane at pH 7.0.Under these conditions, a change in A298 of 2.26 represents the hydrolysis of 1 mol of the substrate. The hydrolysis of the substrate was monitored with a UV/Vis spectrophotometer (Hitachi U 3300).
2.3 Protein concentration
The protein concentration of enzyme solution was determined by A280 or BCA protein assay (Sigma, USA) using bovine serum albumin as a standard. For the homogeneous imidase, 1U of A280 equals 1.17 mg/ml imidase based on the BCA protein assay. Similar results were determined for all the three enzymes studied in this report.
2.4 Protein purification
Imidase taken from pig liver and hydantoinase (bacterial counterpart of imidase) taken from Agrobacterium radiobacter were purified according to the published procedures. Fish liver imidase was purified as described below. Fish (Oreochromis niloticus) were purchased from traditional market. All procedures for protein purification were conducted at 4℃ or in an ice bath. The pH of buffers for enzyme purification refers to the measurements taken at room temperature. An FPLC system (Pharmacia) and a Pharmacia column were used for column chromatography.
Step 1: extract. The imidase was extracted from fresh fish liver (Oreochromis niloticus) (about 200 g) with a 400 ml buffer A (50 mM potassium phosphate, 1 mM PMSF, and 2mM EDTA at pH 8). A fair amount of lipid-like substance was found in the fish liver during the enzyme extraction and removed by filter papers (Whatman, No.1). The suspension was centrifuged at 20,000×g for 1 hr to remove precipitates.
Step 2: salting out. Ammonium sulfate (equivalent to 35% of saturation) was slowly added to the enzyme extract and stirred for 60 min. The suspension was centrifuged at 20,000
×g for 30 min to remove precipitate and an additional ammonium sulfate was added into a supernatant fluid to 60% saturation. After gentle stirring for 60 min, the resultant precipitate was collected by centrifugation (20,000×g, 30min). The precipitate was dissolved in buffer B (10 mM potassium phosphate, 1.5 M ammonium sulfate, 1 mM PMSF, and 2 mM EDTA at pH 7). Then it was stirred gently for 120 min. Insoluble substances were removed by centrifugation (20,000×g, 30 min). The dissolution of the enzyme precipitate was repeated once to ensure that all the imidase activity was collected in the solution.
Step 3: octyl Sepharose. The enzyme solution was applied to a column (4.4×10 cm) of octyl Sepharose CL-4B that had been equilibrated with buffer B followed by washing with 400 ml of the same buffer. The protein was eluted with a reverse linear salt gradient of 1.5–0 M ammonium sulfate using 320 ml each of buffer B and buffer B minus ammonium sulfate.
Active fractions were pooled and desalted by dialysis with buffer C (20 mM potassium phosphate, 1 mM PMSF, and 0.5 M NaCl at pH 7) .
Step 4: chelating Sephacel. The enzyme solution in buffer C was applied to a column (1.6
×10 cm) of chelating Sephacel (fast flow) that had been treated with 1 gel volume of 0.2 M zinc acetate. The column was then equilibrated with buffer C. The loaded column was washed with 150 ml of buffer C, and the enzyme was eluted with a linear glycine gradient from 0 to 1 M with buffer C and buffer C plus 1 M glycine (total volume 240 ml). The active fractions were pooled and desalted by dialysis with buffer D (10 mM potassium phosphate and 1 mM PMSF at pH 7) .
Step 5: hydroxyapatite Sephacel. The imidase solution was loaded into a column (1.6×10 cm) of hydroxyapatite Sephacel that had been equilibrated with buffer D. Imidase was eluted with a linear potassium phosphate gradient from 0.02 to 0.4 M (total in a volume of 300 ml, including 1 mM PMSF at pH 7).
Step 6: Hitrap Q Sepharose. The desalted enzyme solution was loaded into a Hitrap Q column (5 ml, previously equilibrated with buffer D). The enzyme was eluted by buffer D (total volume 25 ml) without a salt gradient. Homogeneous imidase was obtained from active fractions. By the standard assay, the specific activity of fish imidase was 86μmol/min/mg in a typical experiment.
2.5 In gel digestion and identification using LC–ESI–MS
The spots of interest were excised and digested in gel with trypsin according to Shevchenko’s method (Shevchenko et al., 1996). The digestion sample was taken up and analyzed using LC-ESI-MS. The results were correlated with the sequence database using the NCBI and SwissProt database and analyzed by Mascot software.
2.6 RNA extraction and cDNA synthesis
The fish (Tetraodon nigroviridis) were purchased from a pet shop. Fish liver were dissected out and total RNA was extracted with RNeasy® Protect Mini Kit (Qiagen, U.S.A.) following the manufacturer’s protocol and cDNA synthesis was carried out using SuperScript III First-Strand Synthesis System for RT-PCR Kit (Invitrogen, U.S.A.).
2.7 PCR cloning and sequencing
The synthesized cDNA was used as a template for PCR to clone the imidase from fish (Tetraodon nigroviridis). The forward primer was 5'-atggcagaagccggcgagatc-3', and reverse primer was 5'-tcagtcagacgttcccagagcaac-3'. The amplification conditions were 30 cycles of 95 °C for 30 sec, 55 °C for 1 min and 72 °C for 2 min. The PCR product was cloned into pGEM-T easy vector (Promega, U.S.A.) and transformed into DH5α. Plasmid DNA was extracted by alkaline lysis (Premier, Taiwan). The cDNA clone was subjected to sequencing reaction with T7 and SP6 primers and sequenced on an automated DNA sequencer.
2.8 Sequence analysis
The sequence was compared with those in the database using the BlastN algorithm.
Protein sequence alignment and percentage identities were calculated using MuliAlin and ClustalW software.
2.9 Homology Modeling and Molecular Docking
The pair-wise alignment of Agrobacterium radiobacter (Chao et al., 2000) and Burkholderia pickettii hydantoinase sequences are made by ClustalW and revealed a 92.6%
sequence identity. The 3D-structure of Agrobacterium radiobacter hydantoinase was modeled by automatic modeling at the Swiss-Model server (Schwede et al., 2003; Guex and Peitsch, 1997), which the X-ray crystal structure of Burkholderia pickettii hydantoinase (1NFG) was selected as template. A carboxylated lysine was observed in 1NFG, where the modified lysine was conserved in amidohydrolase superfamily. The post modified lysine was manually altered a carboxyl group. Then two zinc ions were added into model structure according to resolved X-ray structure. Fish liver imidase were modeled by automatic modeling at the Swiss-Model server, the X-ray crystal structure of Bacillus Sp. (1YNY) was selected as template. These two model structures were used in GOLD (Jones et al., 1997) as templates to dock parabanic acid and 5-hydantoinacetic acid molecules with 10 genetic algorithm runs.
2.10 Inhibition assay
Non-substrate compounds were examined experimentally as potential inhibitors of imidase. The inhibition assay was done in a similar standard assay condition using 3-iminoisoindoline or phthalimide as substrate. After the inhibitor was mixed with assay solution, the enzyme was added to start the measurement.
2.11 Analysis of Kinetics Data
Results of kinetics experiments were analyzed using nonlinear or linear regression to fit the appropriate equation to the data. The rate constants (Km, Vmax and Ki) were obtained using SigmaPlot 2001, V7.0 and Enzyme Kinetics Module, V1.1 (SPSS Inc., Chicago, IL).
Data used represent mean values derived from three determinations.