Structural Basis for pH-Dependent Oligomerization of Dihydropyrimidinase from Pseudomonas aeruginosa PAO1

Dihydropyrimidinase, a dimetalloenzyme containing a carboxylated lysine within the active site, is a member of the cyclic amidohydrolase family, which also includes allantoinase, dihydroorotase, hydantoinase, and imidase. Unlike all known dihydropyrimidinases, which are tetrameric, pseudomonal dihydropyrimidinase forms a dimer at neutral pH. In this paper, we report the crystal structure of P. aeruginosa dihydropyrimidinase at pH 5.9 (PDB entry 5YKD). The crystals of P. aeruginosa dihydropyrimidinase belonged to space group C2221 with cell dimensions of a = 108.9, b = 155.7, and c = 235.6 Å. The structure of P. aeruginosa dihydropyrimidinase was solved at 2.17 Å resolution. An asymmetric unit of the crystal contained four crystallographically independent P. aeruginosa dihydropyrimidinase monomers. Gel filtration chromatographic analysis of purified P. aeruginosa dihydropyrimidinase revealed a mixture of dimers and tetramers at pH 5.9. Thus, P. aeruginosa dihydropyrimidinase can form a stable tetramer both in the crystalline state and in the solution. Based on sequence analysis and structural comparison of the dimer-dimer interface between P. aeruginosa dihydropyrimidinase and Thermus sp. dihydropyrimidinase, different oligomerization mechanisms are proposed.


Introduction
Dihydropyrimidinase is a key enzyme for pyrimidine catabolism [1,2]. Dihydropyrimidinase catalyzes the reversible cyclization of dihydrouracil to N-carbamoyl-β-alanine in the second step of the pyrimidine degradation pathway (Figure 1). Dihydropyrimidinase can also detoxify xenobiotics with an imide functional group, ranging from linear imides to heterocyclic imides [3][4][5][6][7][8][9]. Homologous enzymes from microorganisms are known as hydantoinase, used as biocatalyst for hydrolysis of 5-monosubstituted hydantoins in the synthesis of D-and L-amino acids [10,11]. Optically pure amino acids have been widely used as intermediates for semisynthesis of antibiotics, active peptides, hormones, antifungal agents, pesticides, and sweeteners. Dihydropyrimidinase and hydantoinase generally possess a similar active site, but their overall sequence identity and substrate speci city may di er [3,12]. For example, hydantoinase puri ed from Agrobacterium species has no 5,6-dihydropyrimidine amidohydrolase activity [13]. Dihydropyrimidinases from the yeast Saccharomyces kluyveri and the slime mold Dictyostelium discoideum do not hydrolyze hydantoin [14]. us, several bacterial hydantoinases are still named and identi ed as dihydropyrimidinase because of their catalytic activity toward natural substrates, namely, dihydrouracil and dihydrothymine. ese bacterial enzymes include Pseudomonas aeruginosa and ermus sp. dihydropyrimidinases [15,16].
Dihydropyrimidinase, hydantoinase, imidase, allantoinase, and dihydroorotase belong to the cyclic amidohydrolase family because of their functional and structural similarities [17]. Members of this enzyme family catalyze the ring-opening hydrolysis of the cyclic amide bond of each substrate in either ve-or six-membered rings. Even if these enzymes have similar functions, they have relatively low amino acid sequence identity. In addition, the substrate selectivity and speci city of these enzymes highly di er [18,19]. Most of the active sites of dihydropyrimidinases, hydantoinases, allantoinases, and dihydroorotases contain four histidines, one aspartate, and one carboxylated lysine residue, which are required for metal binding and catalytic activity [8,15,18,20,21]. e presence of a carboxylated lysine in hydantoinase is also required for the self-assembly of the binuclear metal center [12,20,22] and increases the nucleophilicity of the hydroxide for catalysis [23]. e global architecture of the dihydropyrimidinase monomer consists of two domains, namely, a large domain with a classic (β/α) 8barrel structure core embedding the catalytic dimetal center and a small β-sandwich domain [16,22,24,25].
All known dihydropyrimidinases are tetramers except pseudomonal enzymes. Hydantoinase from P. putida YZ-26 functions as a dimer [26,27]. Recently, we identi ed that dihydropyrimidinase from P. aeruginosa PAO1 also forms a dimer [28]. In addition, the crystal structure of P. aeruginosa PAO1 dihydropyrimidinase indicated that several residues crucial for tetramerization are not found in P. aeruginosa dihydropyrimidinase [28]. In this study, we found that the oligomerization of P. aeruginosa PAO1 dihydropyrimidinase is a pH-dependent process. At pH 5.9, P. aeruginosa PAO1 dihydropyrimidinase mainly formed a tetramer. To con rm this result and determine how this enzyme can also form a tetramer, we also determined the crystal structure of P. aeruginosa PAO1 dihydropyrimidinase at 2.17Å resolution at acidic environment. Structural comparison indicated that although P. aeruginosa PAO1 dihydropyrimidinase can also form a tetramer, the residues being crucial for tetramerization are di erent from those in ermus sp. dihydropyrimidinases.

Cloning, Protein Expression, and Puri cation.
Construction of the P. aeruginosa dihydropyrimidinase expression plasmid has been reported [15]. Recombinant P. aeruginosa dihydropyrimidinase was expressed and puri ed using the protocol described previously [15]. e protein puri ed from the soluble supernatant by Ni 2+ -a nity chromatography (HiTrap HP; GE Healthcare Bio-Sciences, Piscataway, NJ, USA) was eluted with Bu er A (20 mM Tris-HCl, 250 mM imidazole, and 0.5 M NaCl, pH 7.9) and dialyzed against a dialysis bu er (20 mM HEPES and 100 mM NaCl, pH 7.0; Bu er B). Protein purity remained > 97% as determined by SDS-PAGE (Mini-PROTEAN Tetra System; Bio-Rad, CA, USA).

Gel Filtration Chromatography.
Gel ltration chromatography was carried out by the AKTA-FPLC system (GE Healthcare Bio-Sciences, Piscataway, NJ, USA). In brief, puri ed protein (5 mg/mL) in Bu er C (20 mM MES and 100 mM NaCl, pH 5.9) was applied to a Superdex 200 prep grade column (GE Healthcare Bio-Sciences, Piscataway, NJ, USA) equilibrated with the same bu er [29]. e column was operated at a ow rate of 0.5 mL/min, and the proteins were detected at 280 nm. e column was calibrated with proteins of known molecular weight: thyroglobulin (670 kDa), c-globulin (158 kDa), ovalbumin (44 kDa), myoglobin (17 kDa), and vitamin B 12 (1.35 kDa).

Crystallography.
Before crystallization, P. aeruginosa dihydropyrimidinase was concentrated to 20 mg/mL in Bu er C. Crystals were grown at room temperature by hanging drop vapor di usion in 10% PEG 8000, 100 mM HEPES, 200 mM calcium acetate, pH 5.9. Data collection and re nement statistics for the crystal of P. aeruginosa dihydropyrimidinase are shown in Table 1. Data were collected using an ADSC Quantum-315r CCD area detector at SPXF beamline BL13C1 at NSRRC (Taiwan, ROC). All data integration and scaling were carried out using HKL-2000 [30].
ere were four P. aeruginosa dihydropyrimidinase monomers per asymmetric unit. e crystal structure of P. aeruginosa dihydropyrimidinase was solved at 2.17Å resolution with the molecular replacement software AMoRe [31] using the dihydropyrimidinase (PDB entry 5E5C) [28] as  model. After molecular replacement, model building was carried out using XtalView [32]. CNS was used for molecular dynamics re nement [33]. e nal structure was re ned to an R-factor of 0.1759 and an R free of 0.2312. Atomic coordinates and related structural factors have been deposited in the PDB with accession code 5YKD.  1). e unit cell contained eight molecules. An asymmetric unit of the crystal contained four crystallographically independent P. aeruginosa dihydropyrimidinase monomers, in which two zinc ions were found in the active site per monomer (Figure 2(a)). e majority of the electron density for P. aeruginosa dihydropyrimidinase exhibited good quality, and no discontinuity was observed. Brie y, the overall structure of each P. aeruginosa dihydropyrimidinase unit consists of 17 α-helices, 19 β-sheets, and two zinc ions (Figure 2(b)). At pH 5.9, the architecture of the P. aeruginosa dihydropyrimidinase monomer consists of two domains, namely, a large domain with a classic (β/α) 8 -barrel structure core embedding the catalytic dimetal center and a small β-sandwich domain.

Structural
Comparison. e overall structure and architecture of the active site of P. aeruginosa dihydropyrimidinase are similar to those of other dihydropyrimidinases (Figure 3(a)) and other members of the amidohydrolase family of enzymes, such as hydantoinases, dihydroorotases, and allantoinases (Figure 3(b)). e active sites of these enzymes contain four histidines, one aspartate, and one carboxylated lysine residue, which are required for metal binding and catalytic activity [12,14,15,19,20,34,35].

pH-Dependent Oligomerization of P. aeruginosa
Dihydropyrimidinase. It was noted that the crystals of the dimeric P. aeruginosa dihydropyrimidinase belonged to space group P3 1 21 grown at the condition of 28% PEG 6000, 100 mM HEPES, 200 mM lithium acetate, pH 7.5 [28]. Due to the di erent crystallization condition, we attempted to test whether the oligomerization of P. aeruginosa dihydropyrimidinase is pH-dependent. All known dihydropyrimidinases are tetramers. However, pseudomonal dihydropyrimidinase/hydantoinase forms a dimer at neutral pH [26][27][28]. Given that the structure implies that  P. aeruginosa dihydropyrimidinase may also form a tetramer in the crystalline state at pH 5.9 (Figure 2(a)), we performed biochemical veri cation to con rm the oligomerization state. To con rm whether or not the oligomerization of P. aeruginosa dihydropyrimidinase is pH-dependent, we conducted gel ltration chromatography at pH 5.9. As shown in Figure 4, the results revealed that two species with elution volume of 63.25 and 69. 26 mL did coexist. e molecular mass of a P. aeruginosa dihydropyrimidinase monomer, as calculated from the amino acid sequence, is 53 kDa. Assuming that these two forms of P. aeruginosa dihydropyrimidinase have a shape and partial speci c volume similar to the standard proteins, the native molecular masses of P. aeruginosa dihydropyrimidinase were estimated to be 105 and 180 kDa, approximately 1.9 and 3.5 times the molecular mass of a P. aeruginosa dihydropyrimidinase monomer, respectively. In comparison at pH 7.5, gel ltration chromatographic analysis of P. aeruginosa dihydropyrimidinase revealed a single peak; the native molecular mass was estimated to be 117 kDa [28]. e two forms of this enzyme obtained from the gel ltration chromatography at pH 5.9 had similar speci c activity (data not shown). us, P. aeruginosa dihydropyrimidinase did exist as a mixture of dimers and tetramers at pH 5.9.

Structural Insights into Dimer of Dimer (Tetramer)
Formation of Dihydropyrimidinase. In this study, we have identi ed that P. aeruginosa dihydropyrimidinase did exist as a mixture of dimers and tetramers at pH 5.9. To assess how P. aeruginosa dihydropyrimidinase can form a stable tetramer, the dimer-dimer interface was analyzed. In the  crystal of P. aeruginosa dihydropyrimidinase, the four molecules formed two pairs of dimers, B-A and C-D, respectively ( Figure 5). Since the two dimers of P. aeruginosa dihydropyrimidinase associate via few contacts to create the tetramer, it was thought that the tetrameric state may be possibly due to crystal packing forces. We noted that in the crystal, another crystallographically related tetramer B-A-C′-D′ ( Figure 5) was formed and further stabilized via many  hydrogen bonds and salt bridges (Tables 2 and 3). is tetramerization structure was similar to that of ermus sp. dihydropyrimidinase (PDB entry 1GKQ).
We also compared the residues important for tetramerization located at the B-A-C′-D′ dimer-dimer interface with those of ermus sp. dihydropyrimidinase ( Figure 6). Although their overall structures are similar, the important residues for tetramer (dimer B-C′ with dimer A-D′) formation are quite di erent. For the tetramer formation of P. aeruginosa dihydropyrimidinase, many hydrogen bonds with close distance were found: these bonds (<3Å) include ; however, these residues were not found for the tetramer formation of ermus sp. dihydropyrimidinase ( Figure 6). Only A13-D14 hydrogen bond was found in ermus sp. dihydropyrimidinase (i.e., H13-E14 in P. aeruginosa dihydropyrimidinase). us, the dimer-dimer interface between P. aeruginosa dihydropyrimidinase and ermus sp. dihydropyrimidinase was signi cantly di erent (Figure 7). Comparison by superimposition indicated that many Arg residues (R253, R358, R386, R467, and R468) found in P. aeruginosa dihydropyrimidinase, but not in ermus sp. dihydropyrimidinase, may play a crucial role for the pH-dependent oligomerization. If consider the pK a , a much better candidate is His13, which is involved in intermolecular interactions and, dependent on the environment of its side chain, which may easily change protonation state between pH 5.9 and pH 7.5. However, this speculation needs to be con rmed by further biochemical experiments.

Di erent Mechanisms for Tetramer Formation of
Dihydropyrimidinases. In this study, we identi ed P. aeruginosa dihydropyrimidinase can be a tetramer both in the crystalline state and in solution (Figure 4). e structure of the tetrameric ermus sp. dihydropyrimidinase and P. aeruginosa dihydropyrimidinase was compared ( Figure 6). Many important residues for ermus sp. dihydropyrimidinase tetramer formation are di erent from those for P. aeruginosa dihydropyrimidinase (Figure 7). On the basis of these results, we concluded that P. aeruginosa dihydropyrimidinase could form a tetramer, but its oligomerization mechanism di ered from those of other dihydropyrimidinases such as ermus sp. dihydropyrimidinase.

Conflicts of Interest
e authors declare that they have no con icts of interest regarding the publication of this paper.