Crystal structure of dehydroquinate synthase from Thermus thermophilus HB8 showing functional importance of the dimeric state

Abstract

Introduction. Dehydroquinate synthase (DHQS) is a ␤-nicotinamide adenine dinucleotide (NAD ϩ )-dependent metalloenzyme that converts 3-deoxy-D-arabino-heptulosonate-7-phosphate to dehydroquinate in the shikimate pathway of bacteria, microbial eukaryotes, and plants. 1 This enzyme is expected to be one of the targets for novel antifungal and antibacterial drug designs, 2,3 because the shikimate pathway is absent in mammals, and DHQS is required for pathogenic virulence.4 DHQS is known to have a multistep mechanism, where a single active site catalyzes 5 sequential reactions involving alcohol oxidation, phosphate elimination, carbonyl reduction, ring opening, and intramolecular aldol condensation. 5,6 The crystal structure of DHQS from Aspergillus nidulans (AnDHQS) in complex with substrates and metal ions 7,8 elucidated important residues in the active site and ligand-induced intersubunit orientational changes that provide a potential reaction mechanism. The AnDHQS structures also revealed their dimeric state in crystal form, although its biological significance was not fully discussed. This time, we have solved the crystal structure of DHQS from Thermus thermophilus HB8 (TtDHQS) at 1.8 Å resolution, which is the first crystal structure of this enzyme from thermophilic organisms. We show that TtDHQS is present as a homodimer in crystal form, as well as in solution. In this article, we present the structural comparison between AnDHQS and TtDHQS, providing insight into the biological significance of the dimeric state of this enzyme. Materials and Methods. Sample preparation:The expression plasmid pET-11a, which carries a gene encoding TtDHQS (residues 1-348) from the T. thermophilus HB8 genome, 9 was prepared by the RIKEN Structurome Group. Escherichia coli BL21 (DE3) cells were transformed with the expression plasmid and grown at 37°C in LuriaBertani (LB) medium containing 50 g/mL ampicillin for 20 h. The cells were harvested by centrifugation at 6500 rpm for 5 min, suspended in 20 mM Tris-HCl, pH 8.0 (buffer A), containing 0.5 M NaCl and 5 mM 2-mercaptoethanol, and disrupted by sonication. The supernatant was heated at 70°C for 10 min. After the heat treatment, cell debris and denatured proteins were removed by centrifugation (14,000 rpm, 30 min), and the supernatant was used as the crude extract for purification. The crude extract was desalted using a HiPrep 26/10 desalting column (Amersham-Biosciences) and applied onto a SuperQ TOYOPEAL 650 M column (Tosoh) equilibrated with buffer A. Proteins were eluted with a linear gradient of 0 -0.4 M NaCl. After the buffer replacement with 20 mM 2-Morpholinoethanesulfonic acid (MES)-NaOH pH 6.0 (buffer B), the fraction containing TtDHQS was subjected to a RESOURCE S column (Amersham-Biosciences) equilibrated with buffer B, and eluted with a linear gradient of 0 -0.4 M NaCl in buffer B. After the buffer replacement with 10 mM phosphate-NaOH, pH 7.0, the fraction containing TtDHQS was applied onto a Bio-Scale CHT-20-I column (BIO-RAD) equilibrated with the same buffer. Prot...

Structure and Evolution of the Archaeal Lipid Synthesis Enzyme sn-Glycerol-1-phosphate Dehydrogenase

“…A number of these secondary structure-related contractions may also be responsible for the monomeric solution state for MJ G1PDH observed by gel filtration chromatography experiments. MJ G1PDH lacks a number of N-and C-terminal structural elements ascribed to its homologues that enable the formation of biochemically active dimers, octamers, and decamers (33, 37) including an elongated ␤-hairpin loop, which is critical in forming the dimer interface in DHQS from Thermus thermophilus (38). QtPISA analysis (39) of the MJ G1PDH crystal structure indicated a positive protein interaction and free energy gain upon the formation of a MJ G1PDH dimer, revealing some potential for complex formation in solution.…”

Crystal Structure of Glucose-6-Phosphate Isomerase from Thermus thermophilus HB8 Showing a Snapshot of Active Dimeric State

“…In these three conventional GPIs, the dimer interface residues are mostly conserved. The dimerization of conventional GPI is reminiscent of other oligomeric enzymes such as glycine cleavage system Tprotein 45 and dehydroquinate synthase, 46 where also the interface residues are well conserved and the interface area does not depend on the optimum living temperature of the source organism. Therefore, it is likely that the interprotomer arrangement of active site residues is important for the catalytic reaction of the conventional GPIs.…”

Crystal Structure of Novel NADP-dependent 3-Hydroxyisobutyrate Dehydrogenase from Thermus thermophilus HB8

“…This result may indicate the biological importance of these residues in the catalytic function of this protein through maintaining the integrity of the AB dimer, as reported for instance for the dimeric dehydroquinate synthase. 15 The other two interfaces do not contain invariant residues, suggesting that residue conservation is not necessarily required at these interfaces; for instance, they might contribute to the thermostability of proteins, as reported for the dimeric/tetrameric 2-deoxyribose-5-phosphate aldolase. 16 Structural resemblance to other proteins A 3-D structural similarity search using DALI server 17 was performed between the refined model of the HIBADH protomer and the coordinates available in the Protein Data Bank (PDB).…”