Structural Basis for pH-Dependent Oligomerization of Dihydropyrimidinase from <i>Pseudomonas aeruginosa</i> PAO1

Cheng, Jen-Hao; Huang, Chien-Chih; Huang, Yen-Hua; Huang, Cheng-Yang

doi:https://doi.org/10.1155/2018/9564391

Bioinorganic Chemistry and Applications

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Abstract Introduction Materials and Methods Results and Discussion Conflicts of Interest Acknowledgments References Copyright Related Articles

Research Article | Open Access

Volume 2018 | Article ID 9564391 | https://doi.org/10.1155/2018/9564391

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

Jen-Hao Cheng,¹Chien-Chih Huang,¹Yen-Hua Huang,¹and Cheng-Yang Huang^1,2

Academic Editor: Luigi Casella

Received17 Oct 2017

Revised20 Nov 2017

Accepted03 Dec 2017

Published30 Jan 2018

Abstract

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.

1. 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 [3–9]. 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 specificity may differ [3, 12]. For example, hydantoinase purified 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]. Thus, several bacterial hydantoinases are still named and identified as dihydropyrimidinase because of their catalytic activity toward natural substrates, namely, dihydrouracil and dihydrothymine. These bacterial enzymes include Pseudomonas aeruginosa and Thermus 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 five- 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 specificity of these enzymes highly differ [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]. The 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]. The global architecture of the 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 [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 identified 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 confirm this result and determine how this enzyme can also form a tetramer, we also determined the crystal structure of P. aeruginosa PAO1 dihydropyrimidinase at 2.17 Å 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 different from those in Thermus sp. dihydropyrimidinases.

2. Materials and Methods

2.1. Cloning, Protein Expression, and Purification

Construction of the P. aeruginosa dihydropyrimidinase expression plasmid has been reported [15]. Recombinant P. aeruginosa dihydropyrimidinase was expressed and purified using the protocol described previously [15]. The protein purified from the soluble supernatant by Ni²⁺-affinity chromatography (HiTrap HP; GE Healthcare Bio-Sciences, Piscataway, NJ, USA) was eluted with Buffer A (20 mM Tris-HCl, 250 mM imidazole, and 0.5 M NaCl, pH 7.9) and dialyzed against a dialysis buffer (20 mM HEPES and 100 mM NaCl, pH 7.0; Buffer B). Protein purity remained > 97% as determined by SDS-PAGE (Mini-PROTEAN Tetra System; Bio-Rad, CA, USA).

2.2. Gel Filtration Chromatography

Gel filtration chromatography was carried out by the AKTA-FPLC system (GE Healthcare Bio-Sciences, Piscataway, NJ, USA). In brief, purified protein (5 mg/mL) in Buffer 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 buffer [29]. The column was operated at a flow rate of 0.5 mL/min, and the proteins were detected at 280 nm. The column was calibrated with proteins of known molecular weight: thyroglobulin (670 kDa), γ-globulin (158 kDa), ovalbumin (44 kDa), myoglobin (17 kDa), and vitamin B₁₂ (1.35 kDa).

2.3. Crystallography

Before crystallization, P. aeruginosa dihydropyrimidinase was concentrated to 20 mg/mL in Buffer C. Crystals were grown at room temperature by hanging drop vapor diffusion in 10% PEG 8000, 100 mM HEPES, 200 mM calcium acetate, pH 5.9. Data collection and refinement 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]. There were four P. aeruginosa dihydropyrimidinase monomers per asymmetric unit. The crystal structure of P. aeruginosa dihydropyrimidinase was solved at 2.17 Å 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 refinement [33]. The final structure was refined 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.

3. Results and Discussion

3.1. Structure of the P. aeruginosa Dihydropyrimidinase Monomer

Crystals of P. aeruginosa dihydropyrimidinase were grown at room temperature by hanging drop vapor diffusion in 10% PEG 8000, 100 mM HEPES, 200 mM calcium acetate, pH 5.9. The crystals of P. aeruginosa dihydropyrimidinase grown under this condition belonged to space group C222₁ with cell dimensions of a = 108.9, b = 155.7, and c = 235.6 Å. The crystal structure of P. aeruginosa dihydropyrimidinase was solved at 2.17 Å resolution (Table 1). The 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)). The majority of the electron density for P. aeruginosa dihydropyrimidinase exhibited good quality, and no discontinuity was observed. Briefly, 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.

(a)

(b)

3.2. Structural Comparison

The 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)). The 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].

(a)

(b)

Figure 3

Structural comparison. (a) Superposition of the active site of dihydropyrimidinases. Their active sites contain four histidines, one aspartate, and one carboxylated lysine residue, which are required for metal binding and catalytic activity. Dihydropyrimidinases from P. aeruginosa (PDB entry 5E5C; green), Thermus sp. (PDB entry 1GKQ; salmon), Tetraodon nigroviridis (PDB entry 4H01; pale yellow), and the structure (PDB entry 5YKD; purple blue) in this study are shown. The architecture of these active sites is similar. (b) Superposition of the active site of members of the amidohydrolase family. Their active sites contain four histidines, one aspartate, and one carboxylated lysine residue, which are required for metal binding and catalytic activity. P. aeruginosa dihydropyrimidinase (PDB entry 5YKD; purple blue), Escherichia coli allantoinase (PDB entry 3E74; bright orange), Burkholderia pickettii hydantoinase (PDB entry 1NFG; aquamarine), and E. coli dihydroorotase (PDB entry 1J79; brown) are shown. The architecture of these active sites is similar.

3.3. pH-Dependent Oligomerization of P. aeruginosa Dihydropyrimidinase

It was noted that the crystals of the dimeric P. aeruginosa dihydropyrimidinase belonged to space group P3₁21 grown at the condition of 28% PEG 6000, 100 mM HEPES, 200 mM lithium acetate, pH 7.5 [28]. Due to the different 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 [26–28]. 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 verification to confirm the oligomerization state. To confirm whether or not the oligomerization of P. aeruginosa dihydropyrimidinase is pH-dependent, we conducted gel filtration 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. The 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 specific 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 filtration chromatographic analysis of P. aeruginosa dihydropyrimidinase revealed a single peak; the native molecular mass was estimated to be 117 kDa [28]. The two forms of this enzyme obtained from the gel filtration chromatography at pH 5.9 had similar specific activity (data not shown). Thus, P. aeruginosa dihydropyrimidinase did exist as a mixture of dimers and tetramers at pH 5.9.

3.4. Structural Insights into Dimer of Dimer (Tetramer) Formation of Dihydropyrimidinase

In this study, we have identified 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). This tetramerization structure was similar to that of Thermus 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 Thermus 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 different. For the tetramer formation of P. aeruginosa dihydropyrimidinase, many hydrogen bonds with close distance were found: these bonds (<3 Å) include K374(A)–E14(B), H13(A)–E14(B), R386(A)–E14(B), R386(A)–E15(B), R467(A)–V354(B), R468(A)–G357(B), E14(A)–H13(B), E15(A)–R386(B), V354(A)–R467(B), G357(A)–R468(B), H13(C′)–E14(D′), R386(C′)–E15(D′), R468(C′)–G357(D′), E14(C′)–H13(D′), E14(C′)–K374(D′), E15(C′)–R386(D′), and G357(C′)–R468(D′); however, these residues were not found for the tetramer formation of Thermus sp. dihydropyrimidinase (Figure 6). Only A13–D14 hydrogen bond was found in Thermus sp. dihydropyrimidinase (i.e., H13–E14 in P. aeruginosa dihydropyrimidinase). Thus, the dimer-dimer interface between P. aeruginosa dihydropyrimidinase and Thermus sp. dihydropyrimidinase was significantly different (Figure 7). Comparison by superimposition indicated that many Arg residues (R253, R358, R386, R467, and R468) found in P. aeruginosa dihydropyrimidinase, but not in Thermus 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 confirmed by further biochemical experiments.

(a)

(b)

3.5. Different Mechanisms for Tetramer Formation of Dihydropyrimidinases

In this study, we identified P. aeruginosa dihydropyrimidinase can be a tetramer both in the crystalline state and in solution (Figure 4). The structure of the tetrameric Thermus sp. dihydropyrimidinase and P. aeruginosa dihydropyrimidinase was compared (Figure 6). Many important residues for Thermus sp. dihydropyrimidinase tetramer formation are different 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 differed from those of other dihydropyrimidinases such as Thermus sp. dihydropyrimidinase.

Conflicts of Interest

The authors declare that they have no conflicts of interest regarding the publication of this paper.

Acknowledgments

The authors thank the technical services provided by the Synchrotron Radiation Protein Crystallography Facility of the National Core Facility Program for Biotechnology, Ministry of Science and Technology, and the National Synchrotron Radiation Research Center, a national user facility supported by the Ministry of Science and Technology, Taiwan, ROC. This research was supported by a grant from the Ministry of Science and Technology, Taiwan (MOST 106-2320-B-040-004) to Cheng-Yang Huang.

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Copyright © 2018 Jen-Hao Cheng et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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