3.1 Purification of fish liver imidase
Imidase from fish liver (Oreochromis niloticus) was purified about 2630-fold with a 9%
yield (Table 1). A homogeneous protein was obtained as determined by SDS–PAGE (Figure 1). The molecular weight of a single polypeptide chain was estimated to be around 56,000.
3.2 Identification of fish liver imidase
Through in-gel digestion and identification using LC–ESI–MS (Figure 2), several peptide fragments were matched with. As showed in figure 3, the amino acid coverage of human dihydropyrimidinase is 8% and the matched peptide fragments were “-MFMAYK-, -ALGKDDFTK- , -IPNGVNGVEDR-, -MSVIWEK-, -GVHSGKMDENR-”. The amino acid
coverage of putative Canis familiaris dihydropyrimidinase is 12% and the matched peptide fragments were “-MFMAYK-, -EIGAIALVHAENGDLIAEGAK-, -ALGKDDFTK-, -IPNGVNGVER-, -MSVIWEK-, -GVHSGKMDENR-”. The amino acid coverage of mouse dihydropyrimidinase related protein-5 is 4% and the matched peptide fragments were
“-MSVVWER-, -MDENRFVAVTSSNAAK-, -FVAVTSSNAAK-”. Besides, the results were correlated with the sequence database by Mascot software indicated two unnamed proteins, Tetraodon nigroviridis unnamed protein (CAF98068) and Xenopus laevis unnamed protein (gi|54311199), have high amino acid coverage (19% and 11% respectively). Amino acid alignment revealed 73% homology with Homo sapiens dihydropyrimidinase and Tetraodon nigroviridis unnamed protein has the six conserved amino acids in the active site. The condition is the same in the Xenopus laevis unnamed protein (sequence identity is 78%). The detection results of LC–ESI–MS were demonstrated that the purified enzyme is imidase.
3.3 Cloning of fish liver imidase
Total RNA was extracted from Tetraodon nigroviridis liver and cDNA was synthesized by RT with oligo d(T) primer. Base of sequence (CAF98068), a pair of primers, fDHP N-ter (5'-atggcagaagccggcgagatc-3') and fDHP C-ter (5'-tcagtcagacgttcccagagcaac-3’), were designed. After PCR, the full-length cDNA of imidase was obtained. The nucleotide sequence and the deduced amino acid sequence are shown in figure 4. The complete nucleotides of fish imidase contained a 1503 bp open reading frame (ORF) and encoded 500 amino acids. The deduced amino acid sequence of Tetraodon nigroviridis imidase shared relatively high identities with Homo sapiens imidase (NP_001376) and Rattus norvegicus imidase (Q63150).
3.4 Homology Modeling and Generation of the active site structure
Currently, crystal structures of hydantoinase from Thermus sp. (Abendroth et al., 2002), Bacillus stearothermophilus (Cheon et al., 2002), Burkholderia pickettii (Gerlt et al., 2003) and Bacillus sp. AR9 (Radha Kishan et al., 2005) are available. They all consist of a core (β/α)8-barrel fold (Gerlt and Raushel, 2003). A similar fold was also reported in the α-subunits of urease from Klebsiella aerogenes and Bacillus pasteurii, dihydroorotase from E. coli, and the phosphotriesterase from Pseudomonas diminuta ( Jabri et al., 1995; Benini et al., 1999;
Thoden et al., 2001; Vanhooke et al., 1996). In all those structures, six highly conserved metal binding residues, four histidines, one carboxylated lysine, and one aspartic acid were coordinated with two metal ions to stabilize the orientation of the binuclear center. According to the crystallized structures with bound inhibitors in urease, dihydroorotase, and phosphotriesterase, the active site of hydantoinase could be expected to locate in this region.
In hydantoinase, allantoinase and dihydroorotase, the two divalent metal ions are presented.
The carboxylateded lysine residue bridges the two metal ions. However, in mammalian
imidase and fish liver imidase, they function with single divalent metal ion in the active site.
The mystery about the different number of metals in the different species has been unclear all the time. Surprisingly, Aquifex aeolicus dihydroorotase has recently been reported that also has single divalent metal ions in the active site, and the metal site was correlated to theα- zinc site in E. coli dihydroorotase and bacterial hydantoinase.
To build up the active site of hydantoinase from Agrobacterium tumefaciens and fish liver imidase from Tetraodon nigroviridis performed in this study, the crystal structure of Burkholderia pickettii hydantoinase (PDB code 1NFG.) and Bacillus sp. AR 9 (PDB code 1YNY) were used as template respectively. The sequence identity of the Agrobacterium tumefaciens hydantoinase and Burkholderia pickettii hydantoinas is 92.6% (Figure 5). In the structure of Agrobacterium tumefaciens hydantoinase (Figure 7b), the active site that contains binuclear centre is located at one end of the TIM barrel distant from the β-rich domain at the end of a roughly 15 Å deep hydrophobic cleft. The metal binding residues are H57, H59, Kcx148, H181, H237, and D313. α- zinc is coordinated by H57 Nε2, H59 Nε2, Kcx148 Ox2 and D313 Oδ1. β-zinc is coordinated by Kcx148 Ox1, H181 Nδ1, and H237 Nε2.
The active site of Tetraodon nigroviridis imidase was modeled used Bacillus sp. AR 9 (PDB code 1YNY) as template. The sequence identity of the Tetraodon nigroviridis imidase and Bacillus sp. hydantoinas is 42% (Figure 6). In previous study, biochemical analysis of mammalian and fish (Oreochromis niloticus) liver imidase found one tightly bound zinc atom per molecule but its specific binding site, α or β, was unknown. D321, a conserved catalytic residue, is 2.45 Å from Znα and 4.77 Å from Znβ in model structure. Aquifex aeolicus dihydroorotase structure has only one metal ion in α- site. So one zinc ion was added into modeled structure in the α- site coordinated by H62 Nε2, H64 Nε2, D321 Oδ1 and HOH1.
Comparing structures of Tetraodon nigroviridis imidase with Agrobacterium tumefaciens hydantoinase, the side chain of K154 without post modification in fish imidase was shorter
than Kcx148 in hydantoinase. K154 in fish imidase swings out the active site (Figure 7a) and away from α- zinc.
3.5 Molecular Docking
In our previous study, molecular docking selected two compounds, 5-hydantoinacetic acid and parabanic acid, can enter the active site of Agrobacterium tumefaciens hydantoinase and have a reasonable orientation for reaction. Due to the metal contents in the various species were different, we furthermore docked 5-hydantoinacetic acid (Figure 8) and parabanic acid (Figure 9) into Tetraodon nigroviridis imidase by GOLD. Interestingly, both of them have different orientations than in Agrobacterium tumefaciens hydantoinase.
Computational results were confirmed by a series enzymatic assays. The substrate activities were measured at 25℃ in 100 mM Bis-Tris propane at pH 7 and using parabanic acid or 5-hydantoinacetic acid as substrate. Substrate activity results (Table 2) showed that Agrobacterium tumefaciens hydantoinase did hydrolyze the 5-hydantoinacetic acid and parabanic acid but non determined in Oreochromis niloticus imidase. Moreover, inhibition assay (Table 3) showed 5-hydantoinacetic acid and parabanic acid were not substrates in Oreochromis niloticus fish imidase, but competitive inhibitors. Pig liver imidase with one tightly bound zinc atom per molecule also had identical behavior with Oreochromis niloticus imidase.
A reasonable explanation is that the open location and free H181 and H237 in Tetraodon nigroviridis imidase will form different active site with Agrobacterium tumefaciens hydantoinase. Molecular docking of 5-hydantoinacetic acid into Agrobacterium tumefaciens hydantoinase showed it can enter the active site, and N2 of 5-hydantoinacetic acid lay within hydrogen bonding distance of the carbonyl oxygen of T286 (2.73 Å), O3 form a hydrogen bond with peptic NH of T286 (2.86 Å) and peptic NH of N335 had a distance of 3.43 Å with
O4 of the 5-hydantoinacetic acid. However in Tetraodon nigroviridis imidase structure, the β-zinc site is open and GOLD docking result showed that 5-hydantoinacetic acid can enter the active site, but the imide ring was swung out and away from the metal. For Tetraodon nigroviridis imidase, 5-hydantoinacetic acid is not the substrate, but a competitive inhibitor.
GOLD docking of parabanic acid into Agrobacterium tumefaciens hydantoinase also showed it can enter the active site, with N2 of parabanic acid hydrogen bonding with carbonyl oxygen of T286 (2.91 Å), O3 forming a hydrogen bond with peptic NH of T286 (2.86 Å) and peptic NH of N335 had a distance of 3.35 Å with O4 of the parabanic acid. GOLD docking with parabanic acid into Tetraodon nigroviridis imidase showed it tended to the open location and H181 and H237 equal to the β-zinc ligands in hydantoinase can interact with parabanic acid by forming hydrogen bond. Peptic NH of H187 had a distance of 3.45 Å with O3 of the parabanic acid, O1 form a hydrogen bond with peptic NH of H243 (4.25 Å) and peptic NH of K154 had a distance of 3.01 Å with O5 of the parabanic acid. Parabanic acid can enter the active site and form weak hydrogen bonds; maybe this is the reason parabanic acid is not a strong competitive inhibitor for Tetraodon nigroviridis imidase.
3.6 Proposed catalytic mechanism of fish liver imidase
Fish liver imidase would contain a mononuclear metal center within the active site. It is proposed that metal ion in imidase is to coordinate substrate and maintain a suitable active site.
The α-zinc ion is bound by His62, His64, Asp321 and HOH. Nucleophilic attack by the hydroxide is assisted by general base catalysis from the side chain carboxylate of Asp321.
Then the intermediate is stabilized by ligating to metal ion. Collapse of this intermediate and cleavage of the carbon-nitrogen bond are assisted by the simultaneous protonation of the amide nitrogen by Asp321. The metal ion stabilized the developing negative charge of imide substrates.
4. Conclusions
The mystery about the different number of metals in the different species has been unclear all the time. In this study, the sequence of Tetraodon nigroviridis imidase was obtained. Then homology modeling and molecular docking were used to understand this question. The Tetraodon nigroviridis imidase structure exhibit three major differences with Agrobacterium tumefaciens hydantoinase: (1) they have zinc only in α- site coordinated by H62 Nε2, H64 Nε2, D321 Oδ1 and HOH1. (2) they have open β- site and free H187 and H243. (3) side chain of K154 without post modification was shorter than Kcx148 in hydantoinase. Molecular docking of 5-hydantoinacetic acid and parabanic acid into Tetraodon nigroviridis imidase showed that both of them can enter the active site, but had different orientations than in Agrobacterium tumefaciens hydantoinase. Enzymatic assays showed that Agrobacterium tumefaciens hydantoinase did hydrolyze the 5-hydantoinacetic acid and parabanic acid. For Tetraodon nigroviridis imidase, 5-hydantoinacetic acid and parabanic acid are not the substrates, but competitive inhibitors. A reasonable explanation is that compounds tended to the open β- site and formed weak hydrogen bonds with free residues, K154, H187 and H243.
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Table 1. Summary of purification of imidase from fish liver (Oreochromis niloticus) a
Step Volume Total Activity Total Protein Specific Activity Yield Fold of (ml) (μmol/min) (mg) (μmol/min/mg) (%) purification
Extract 370 1180.4 35768 0.03 100 1
35% Salt out 360 1006.2 17054 0.06 85 1.8
60% Salt out 50 873.5 6065 0.14 74 4.4
Octyl Sepharose 15 501.9 529.5 0.95 43 28.7
Chelating Sephacel 10 297.4 15.4 19.3 25 586
Hydroxyapatite 2 153.1 2.82 54 13 1642
HiTrap Q Sepharose 0.5 106.1 1.22 87 9 2630
a All procedures for purification of fish liver imidase were described in “2.4 protein purification” and the assay was made according to
“2.2 enzyme standard assay”.
Figure 1. The purity of fish liver (Oreochromis niloticus) imidase.
SDS-PAGE (12%) of purified fish liver imidase (56K). Lane 1, protein standard (from Pharmacia) with size noted in; lane 2, extract; lane 3, 35% salt out; lane 4, 60% salt out; lane 5, Octyl Sepharose; lane 6, Chelating Sephacel; lane 7, Hydroxyapatite; lane 8, HiTrap Q Sepharose.
a b
c
Score Sequence Coverage Accession No. Definition
Score Sequence Coverage Accession No. Definition