Crystal structure of the type II 3-dehydroquinase from Helicobacter pylori


  • Byung Il Lee,

    1. Laboratory of Structural Proteomics, School of Chemistry and Molecular Engineering, Seoul National University, Seoul, Korea
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  • Je Eun Kwak,

    1. Laboratory of Structural Proteomics, School of Chemistry and Molecular Engineering, Seoul National University, Seoul, Korea
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  • Se Won Suh

    Corresponding author
    1. Laboratory of Structural Proteomics, School of Chemistry and Molecular Engineering, Seoul National University, Seoul, Korea
    • School of Chemistry and Molecular Engineering, Seoul National University, Seoul 151-742, Korea
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Helicobacter pylori is a spiral-shaped, Gram-negative bacterium that lives in the stomach and duodenum. H. pylori infection is associated with peptic ulcer disease, chronic gastritis, mucosa-associated lymphoid tissue lymphoma, and gastric adenocarcinoma. The enzyme 3-dehydroquinate dehydratase or 3-dehydroquinase (DHQase; EC, which catalyzes the interconversion of 3-dehydroquinate and 3-dehydroshikimate, is an attractive target for developing antibacterial compounds specific for H. pylori. DHQases fall into two groups: type I and type II. They have different biochemical and biophysical properties, and no sequence similarity exists between them.1 Type I enzymes are generally found in the biosynthetic shikimate pathway and use a Schiff base intermediate formed at the conserved lysine residue, and catalyze the elimination of water with syn stereochemistry.2, 3 They have subunit molecular masses of ∼25 kDa, form dimers in the case of monofuntional enzymes such as Escherichia coli type I DHQase, and are thermally labile.3 Type II enzymes serve either the biosynthetic shikimate pathway or catabolic quinate pathway,1 or both.4 They have smaller subunit molecular masses than type I enzymes (16–18 kDa), oligomerize into dodecamers of ∼200 kDa, and are heat stable. H. pylori has only the type II enzyme (167 residues, 18,483 Da), in contrast with gut organisms such as E. coli, which have type I enzymes only. Crystal structures of type II DHQases from Mycobacterium tuberculosis5 and Streptomyces coelicolor,6 as well as type I DHQase from Salmonella typhi,5 have been reported. The type II DHQases from M. tuberculosis (147 residues) and S. coelicolor (157 residues) show 34.7% and 37.7% sequence identity, respectively, to that from H. pylori over the entire polypeptide chain. There are subtle differences among the type II DHQases, as indicated by the discrimination between different type II enzymes by rationally designed inhibitors7 and different interactions of type II DHQases from M. tuberculosis and S. coelicolor with phosphate and sulfate.8 Therefore, structural information on DHQase from H. pylori will be valuable for structure-based design of selective inhibitors against H. pylori. Here, we present its crystal structure, which reveals an electron density for a ligand bound in the active site.

Materials and Methods.

Overexpression of H. pylori type II DHQase in the intact form, its crystallization, and X-ray data collection have been reported elsewhere.9 The structure was solved by the molecular replacement method with the use of the structure of M. tuberculosis DHQase [Protein Data Bank (PDB) code 2DHQ] as a search model. The refined model (PDB code 1J2Y) consists of 1,221 nonhydrogen atoms from 158 amino acid residues (residues 1–158) in a monomer, 19 water molecules, and one 3-dehydroquinate molecule in the asymmetric unit. The crystallographic R/Rfree values are 21.8%/27.2% for reflections with I > 2σ in the resolution range 20.0–2.6 Å. Refinement statistics are shown in Table I. Of 136 nonglycine and nonproline residues, 86.0% are in the most favored regions of the Ramachandran plot, 12.5% in additionally allowed regions, and 1.5% in generously allowed regions. Compared with most other type II DHQases, H. pylori DHQase is longer at its C-terminus by ∼13–21 residues. Part of this C-terminal extension (residues 159–167) does not have an electron density, presumably because it is disordered in the crystal. A truncated form (residues 1–147) of H. pylori type II DHQase has also been overexpressed and crystallized, with the hope that it would result in better diffracting crystals. However, the crystals diffracted to ∼3.4 Å resolution only, and the reflection spots were split. Thus, the truncated form was not pursued further.

Table I. Refinement Statistics
  • a

    Values in parentheses refer to the highest resolution shell (2.76–2.6 Å).

  • b

    Rfree is calculated from the randomly selected 10% set of reflections not included in the calculation of the R factor.

Resolution range (Å)20–2.6
No. of reflections used5133 (642)a
No. of nonhydrogen protein atoms1221
No. of water molecules19
No. of hetero atoms (dehydroquinate)13
R/Rfreeb (%)21.8 (33.8)a/27.2 (39.7)a
RMSD from ideal geometry 
 Bond length (Å)0.009
 Bond angle (°)1.4
Average B factor (Å2) 
 Protein atoms46.2
 Water molecules44.5
 Hetero atoms (dehydroquinate)39.0

Results and Discussion.

H. pylori DHQase forms a dodecamer of 23 symmetry, with each of the four trimers occupying the face of a tetrahedron [Fig. 1(A)]. Each subunit has a flavodoxin-like fold, comprised of a central five-stranded parallel β-sheet, which is flanked by two α-helices (α1 and α5) on one side and three α-helices (α2, α3, and α4) on the other side [Fig. 1(B)]. This is the same fold as that seen in M. tuberculosis and S. coelicolor enzymes. The root-mean-square difference between M. tuberculosis (PDB code 2DHQ) and H. pylori enzymes is 1.21 Å for 126 Cα atom pairs, and that between S. coelicolor (PDB code 1GU1; A chain) and H. pylori enzymes is 1.84 Å for 129 Cα atom pairs, respectively.

Figure 1.

Structure of H. pyloritype II DHQase. A: Dodecameric architecture. B: Monomer fold. C: Stereo view of the active site with a molecule of dehydroquinate (in red). The Cα backbone of one subunit is colored green, whereas that of the neighboring subunit is pink. Sidechains of 10 key residues are colored blue or cyan.

In the structure of H. pylori DHQase, the active-site lid domain (residues 16–23) is in the closed conformation, similar to the 2,3-anhydro-quinic acid complex or the phosphate complex of S. coelicolor DHQase. In the apo structure of S. coelicolor DHQase, the lid domain is more open. The lid domain of H. pylori DHQase contains two key residues, Arg17 and Tyr22. Corresponding residues of M. tuberculosis DHQase have been found to be essential for enzyme activity.10 However, in M. tuberculosis DHQase, only a weak electron density defines poorly the flexible loop consisting of residues Arg19–Tyr24.

An extra electron density was identified in the active site of H. pylori DHQase, although we did not include any ligand in the crystallization medium. The electron density was modeled as the substrate dehydroquinate in the absence of information about its identity. Ten key residues in the active site are shown in Figure 1(C). The orientation of the substrate and the arrangement of the key residues in the active site are highly similar to the 2,3-anhydro-quinic acid complex of S. coelicolor DHQase.6 Thus, it is possible to assign likely roles of key residues by analogy. Tyr22(Tyr28) abstracts a proton by acting as a base, and its pKa value is modulated by the charged sidechains of Arg17(Arg23) and Arg109(Arg113). The corresponding residues of S. coelicolor DHQase are given in parentheses. Asp89*(Asp92*) and Arg113(Arg117) form an ion pair, and their effects on Tyr22(Tyr28) may be canceled out. An asterisk after the residue number denotes that the residue comes from a neighboring subunit. Arg23 in the 2,3-anhydro-quinic acid complex of S. coelicolor DHQase is displaced away from the sidechain of Tyr 28 by the bound molecules of glycerol and tartrate. His82(His85) is close to the C5 hydroxyl group of the substrate. His102(His106) acts as a general acid for donating a proton to the C1 hydroxyl group, which is held in the proper position by interacting with Asn76(Asn79). The residue corresponding to Thr104 in H. pylori DHQase is Ser in all other type II DHQases.


We thank Professor N. Sakabe and his staff for assistance during data collection at beamline BL-18B of Photon Factory, Japan.