Three crystal structures of the molybdenum-cofactor biosynthesis protein MogA from two highly thermophilic organisms have been determined at high resolution. Comparative analyses revealed the residues involved in oligomerization. In addition, molecular-dynamics and docking studies suggested the binding affinities of several small molecules towards MogA and homologous proteins.

Molybdenum-cofactor (Moco) biosynthesis is an evolutionarily conserved pathway in almost all kingdoms of life, including humans. Two proteins, MogA and MoeA, catalyze the last step of this pathway in bacteria, whereas a single two-domain protein carries out catalysis in eukaryotes. Here, three crystal structures of the Moco-biosynthesis protein MogA from the two thermophilic organisms Thermus thermophilus (TtMogA; 1.64 Å resolution, space group P21) and Aquifex aeolicus (AaMogA; 1.70 Å resolution, space group P21 and 1.90 Å resolution, space group P1) have been determined. The functional roles and the residues involved in oligomerization of the protein molecules have been identified based on a comparative analysis of these structures with those of homologous proteins. Furthermore, functional roles have been proposed for the N- and C-terminal residues. In addition, a possible protein–protein complex of MogA and MoeA has been proposed and the residues involved in protein–protein interactions are discussed. Several invariant water molecules and those present at the subunit interfaces have been identified and their possible structural and/or functional roles are described in brief. In addition, molecular-dynamics and docking studies with several small molecules (including the substrate and the product) have been carried out in order to estimate their binding affinities towards AaMogA and TtMogA. The results obtained are further compared with those obtained for homologous eukaryotic proteins.

GenX, a lysyl-tRNA synthetase paralogue from Escherichia coli, has been overexpressed in E. coli, purified by three chromatographic steps and cocrystallized with a lysyl adenylate analogue (LysAMS) by the hanging-drop vapour-diffusion method using PEG 4000 as a precipitant.

GenX, a lysyl-tRNA synthetase paralogue from Escherichia coli, was overexpressed in E. coli, purified by three chromatographic steps and cocrystallized with a lysyl adenylate analogue (LysAMS) by the hanging-drop vapour-diffusion method using PEG 4000 as a precipitant. The GenX–LysAMS crystals belonged to the triclinic space group P1, with unit-cell parameters a = 54.80, b = 69.15, c = 94.08 Å, α = 95.47, β = 106.51, γ = 90.46°, and diffracted to 1.9 Å resolution. Furthermore, GenX was cocrystallized with translation elongation factor P (EF-­P), which is believed to be a putative substrate of GenX, and LysAMS using PEG 4000 and ammonium sulfate as precipitants. The GenX–EF-P–LysAMS crystals belonged to the monoclinic space group P21, with unit-cell parameters a = 105.93, b = 102.96, c = 119.94 Å, β = 99.4°, and diffracted to 2.5 Å resolution. Structure determination of the E. coli GenX–LysAMS and GenX–EF-P–LysAMS complexes by molecular replacement was successful and structure refinements are now in progress.

SAICAR synthetase from P. horikoshii OT3 has been cloned, expressed, purified and crystallized.

The study of proteins involved in de novo biosynthesis of purine nucleotides is central in the development of antibiotics and anticancer drugs. In view of this, a protein from the hyperthermophile Pyrococcus horikoshii OT3 was isolated, purified and crystallized using the microbatch method. Its primary structure was found to be similar to that of SAICAR synthetase, which catalyses the seventh step of de novo purine biosynthesis. A diffraction-quality crystal was obtained using Hampton Research Crystal Screen II condition No. 34, consisting of 0.05 M cadmium sulfate hydrate, 0.1 M HEPES buffer pH 7.5 and 1.0 M sodium acetate trihydrate, with 40%(v/v) 1,4-butanediol as an additive. The crystal belonged to space group P31, with unit-cell parameters a = b = 95.62, c = 149.13 Å. Assuming the presence of a hexamer in the asymmetric unit resulted in a Matthews coefficient (V M) of 2.3 Å3 Da−1, corresponding to a solvent content of about 46%. A detailed study of this protein will yield insights into structural stability at high temperatures and should be highly relevant to the development of antibiotics and anticancer drugs targeting the biosynthesis of purine nucleotides.

1,3-Propanediol dehydrogenase (Aq_1145) from A. aeolicus VF5 has been overexpressed, purified and crystallized. The crystals diffracted to 2.4 Å resolution.

1,3-Propanediol dehydrogenase is an enzyme that catalyzes the oxidation of 1,3-­propanediol to 3-hydroxypropanal with the simultaneous reduction of NADP+ to NADPH. SeMet-labelled 1,3-propanediol dehydrogenase protein from the hyperthermophilic bacterium Aquifex aeolicus VF5 was overexpressed in Escherichia coli and purified to homogeneity. Crystals of this protein were grown from an acidic buffer with ammonium sulfate as the precipitant. Single-wavelength data were collected at the selenium peak to a resolution of 2.4 Å. The crystal belonged to space group P32, with unit-cell parameters a = b = 142.19, c = 123.34 Å. The structure contained two dimers in the asymmetric unit and was solved by the MR-SAD approach.

To elucidate which RNA polymerase structural state a particular T. thermophilus Gre-family protein (Gfh1) associates with, the T. thermophilus RNAP elongation complex was cocrystallized with Gfh1.

RNA polymerase (RNAP) elongates RNA by iterative nucleotide-addition cycles (NAC). A specific structural state (or states) of RNAP may be the target of transcription elongation factors. Gfh1, a Thermus thermophilus Gre-family protein, inhibits NAC. To elucidate which RNAP structural state Gfh1 associates with, the T. thermophilus RNAP elongation complex (EC) was cocrystallized with Gfh1. Of the 70 DNA/RNA scaffolds tested, two (for EC1 and EC2) were successfully crystallized. In the presence of Gfh1, EC1 and EC2 yielded crystals belonging to space group P21 with similar unit-cell parameters (crystals 1 and 2, respectively). X-ray diffraction data sets were obtained at 3.6 and 3.8 Å resolution, respectively.

The structure of a protein involved in the molybdopterin and molybdenum co-factor biosynthesis pathways of Sulfolobus tokodaii has been solved to a resolution of 1.9 Å.

The structure of a probable Mo-cofactor biosynthesis protein B from Sulfolobus tokodaii, belonging to space group P6422 with unit-cell parameters a = b = 136.68, c = 210.52 Å, was solved by molecular replacement to a resolution of 1.9 Å and refined to an R factor and R free of 16.8% and 18.5%, respectively. The asymmetric unit contains a trimer, while the biologically significant oligomer is predicted to be a hexamer by size-exclusion chromatography. The subunit structure and fold of ST2315 are similar to those of other enzymes that are known to be involved in the molybdopterin- and molybdenum cofactor-biosynthesis pathways.

The structure of the stationary phase survival protein SurE protein from the hyperthermophile Aquifex aeolicus has been solved to 1.5 Å resolution. The divalent-metal-ion-dependent phosphatase active-site pocket is occupied by sulfate ions from the crystallization medium.

SurE is a stationary-phase survival protein found in bacteria, eukaryotes and archaea that exhibits a divalent-metal-ion-dependent phosphatase activity and acts as a nucleotidase and polyphosphate phosphohydrolase. The structure of the SurE protein from the hyperthermophile Aquifex aeolicus has been solved at 1.5 Å resolution using molecular replacement with one dimer in the asymmetric unit and refined to an R factor of 15.6%. The crystal packing reveals that two dimers assemble to form a tetramer, although gel-filtration chromatography showed the presence of only a dimer in solution. The phosphatase active-site pocket was occupied by sulfate ions from the crystallization medium.

The structure of d-lactate dehydrogenase from Aquifex aeolicus has been determined with each subunit of the homodimer in a ‘closed’ conformation and with the NAD+ cofactor and lactate (or pyruvate) bound at the inter-domain active-site cleft.

The crystal structure of d-lactate dehydrogenase from Aquifex aeolicus (aq_727) was determined to 2.12 Å resolution in space group P212121, with unit-cell parameters a = 90.94, b = 94.43, c = 188.85 Å. The structure was solved by molecular replacement using the coenzyme-binding domain of Lactobacillus helveticus d-lactate dehydrogenase and contained two homodimers in the asymmetric unit. Each subunit of the homodimer was found to be in a ‘closed’ conformation with the NADH cofactor bound to the coenzyme-binding domain and with a lactate (or pyruvate) molecule bound at the interdomain active-site cleft.

The structure of ribose-5-phosphate isomerase from Methanocaldococcus jannaschii has been solved to 1.78 Å resolution, with the active site occupied by two molecules of propylene glycol mimicking the binding of a known arabinose-5-phosphate inhibitor.

Ribose-5-phosphate isomerase is a ubiquitous intracellular enzyme of bacterial, plant and animal origin that is involved in the pentose phosphate cycle, an essential component of cellular carbohydrate metabolism. Specifically, the enzyme catalyses the reversible conversion of ribose 5-phosphate to ribulose 5-­phosphate. The structure of ribose-5-phosphate isomerase from Methano­caldococcus jannaschii has been solved in space group P21 to 1.78 Å resolution using molecular replacement with one homotetramer in the asymmetric unit and refined to an R factor of 14.8%. The active site in each subunit was occupied by two molecules of propylene glycol in different orientations, one of which corresponds to the location of the phosphate moiety and the other to the location of the furanose ring of the inhibitor.

The structure of a putative β-phosphoglucomutase from Thermotoga maritima belonging to the haloacid dehalogenase (HAD) hydrolase family has been determined to 1.74 Å resolution.

The structure of TM1254, a putative β-phosphoglucomutase from T. maritima, was determined to 1.74 Å resolution in a high-throughput structural genomics programme. Diffraction data were obtained from crystals belonging to space group P22121, with unit-cell parameters a = 48.16, b = 66.70, c = 83.80 Å, and were refined to an R factor of 19.2%. The asymmetric unit contained one protein molecule which is comprised of two domains. Structural homologues were found from protein databases that confirmed a strong resemblance between TM1254 and members of the haloacid dehalogenase (HAD) hydrolase family.

The crystal structure of dihydrodipicolinate synthase from the (S)-lysine synthesis pathway of Methanocaldococcus jannaschii has been solved to 2.2 Å resolution, revealing a functional homotetramer.

In bacteria and plants, dihydrodipicolinate synthase (DHDPS) plays a key role in the (S)-lysine biosynthesis pathway. DHDPS catalyzes the first step of the condensation of (S)-aspartate-β-semialdehyde and pyruvate to form an unstable compound, (4S)-4-hydroxy-2,3,4,5-tetrahydro-(2S)-dipicolinic acid. The activity of DHDPS is allosterically regulated by (S)-lysine, a feedback inhibitor. The crystal structure of DHDPS from Methanocaldococcus jannaschii (MjDHDPS) was solved by the molecular-replacement method and was refined to 2.2 Å resolution. The structure revealed that MjDHDPS forms a functional homo­tetramer, as also observed in Escherichia coli DHDPS, Thermotoga maritima DHDPS and Bacillus anthracis DHDPS. The binding-site region of MjDHDPS is essentially similar to those found in other known DHDPS structures.

The putative 4-amino-4-deoxychorismate lyase (TTHA0621) from T. thermophilus HB8 was cloned, overexpressed, purified and crystallized. Its crystal structure was determined by a combination of SAD and molecular-replacement methods and was refined to 1.93 Å resolution.

The pyridoxal 5′-phosphate-dependent enzyme 4-amino-4-deoxychorismate lyase converts 4-amino-4-deoxychorismate to p-aminobenzoate and pyruvate in one of the crucial steps in the folate-biosynthesis pathway. The primary structure of the hypothetical protein TTHA0621 from Thermus thermophilus HB8 suggests that TTHA0621 is a putative 4-amino-4-deoxychorismate lyase. Here, the crystal structure of TTHA0621 is reported at 1.93 Å resolution. The asymmetric unit contained four NCS molecules related by 222 noncrystallographic symmetry, in which the formation of intact dimers may be functionally important. The cofactor pyridoxal 5′-phosphate (PLP) binds to the protein in the large cleft formed by the N-terminal and C-terminal domains of TTHA0621. The high structural similarity and the conservation of the functional residues in the catalytic region compared with 4-amino-4-deoxychorismate lyase (PabC; EC 4.1.3.38) from Escherichia coli suggest that the TTHA0621 protein may also possess 4-amino-4-deoxychorismate lyase activity.

The structure of glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic archaeon Methanocaldococcus jannaschii was determined to 1.81 Å resolution with the NADP+ cofactor at the nucleotide binding site.

The X-ray crystal structure of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from the hyperthermophilic archaeon Methanocaldococcus jannaschii (Mj-GAPDH) was determined to 1.81 Å resolution. The crystal belonged to space group C2221, with unit-cell parameters a = 83.4, b = 152.0, c = 118.6 Å. The structure was solved by molecular replacement and was refined to a final R factor of 17.1% (R free = 19.8%). The final structure included the cofactor NADP+ at the nucleotide-binding site and featured unoccupied inorganic and substrate phosphate-binding sites. A comparison with GAPDH structures from mesophilic sources suggested that Mj-GAPDH is stabilized by extensive electrostatic interactions between the C-terminal α-helices and various distal loop regions, which are likely to contribute to thermal stability. The key phosphate-binding residues in the active site of Mj-GAPDH are conserved in other archaeal GAPDH proteins. These residues undergo a conformational shift in response to occupancy of the inorganic phosphate site.

The first crystal structure of 4-methyl-5-β-hydroxyethylthiazole kinase from an archaeon (P. horikoshii OT3) has been determined at 1.85 Å resolution. Comparative analyses of sequences and structures and modelling studies are presented.

4-Methyl-5-β-hydroxyethylthiazole kinase (ThiK) catalyses the phosphorylation of the hydroxyl group of 4-methyl-5-β-hydroxyethylthiazole. This work reports the first crystal structure of an archaeal ThiK: that from Pyrococcus horikoshii OT3 (PhThiK) at 1.85 Å resolution with a phosphate ion occupying the position of the β-phosphate of the nucleotide. The topology of this enzyme shows the typical ribokinase fold of an α/β protein. The overall structure of PhThiK is similar to those of Bacillus subtilis ThiK (BsThiK) and Enterococcus faecalis V583 ThiK (EfThiK). Sequence analysis of ThiK enzymes from various sources indicated that three-quarters of the residues involved in interfacial regions are conserved. It also revealed that the amino-acid residues in the nucleotide-binding, magnesium ion-binding and substrate-binding sites are conserved. Binding of the nucleotide and substrate to the ThiK enzyme do not influence the quaternary association (trimer) as revealed by the crystal structure of PhThiK.

The putative ABC transporter ATP-binding protein TM0222 from T. maritima was cloned, overproduced, purified and crystallized. A complete MAD diffraction data set has been collected to 2.3 Å resolution.

Adenosine triphosphate (ATP) binding cassette transporters (ABC transporters) are ATP hydrolysis-dependent transmembrane transporters. Here, the overproduction, purification and crystallization of the putative ABC transporter ATP-binding protein TM0222 from Thermotoga maritima are reported. The protein was crystallized in the hexagonal space group P6422, with unit-cell parameters a = b = 148.49, c = 106.96 Å, γ = 120.0°. Assuming the presence of two molecules in the asymmetric unit, the calculated V M is 2.84 Å3 Da−1, which corresponds to a solvent content of 56.6%. A three-wavelength MAD data set was collected to 2.3 Å resolution from SeMet-substituted TM0222 crystals. Data sets were collected on the BL38B1 beamline at SPring-8, Japan.

The crystal structure of an N-terminally (25 residues) truncated fragment of selenophosphate synthetase (SPS-ΔN) from Aquifex aeolicus has been determined at 2.0 Å resolution.

Selenophosphate synthetase (SPS) catalyzes the activation of selenide with ATP to synthesize selenophosphate, the reactive selenium donor for biosyntheses of both the 21st amino acid selenocysteine and 2-selenouridine nucleotides in tRNA anticodons. The crystal structure of an N-terminally (25 residues) truncated fragment of SPS (SPS-ΔN) from Aquifex aeolicus has been determined at 2.0 Å resolution. The structure revealed SPS to be a two-domain α/β protein, with domain folds that are homologous to those of PurM-superfamily proteins. In the crystal, six monomers of SPS-ΔN form a hexamer of 204 kDa, whereas the molecular weight estimated by ultracentrifugation was ∼63 kDa, which is comparable to the calculated weight of the dimer (68 kDa).

Three conventional robots were subjected to a crystallization screening test involving 18 proteins from T. thermophilus HB8 using the sitting- and hanging-drop vapour-diffusion and microbatch methods. The number of diffraction-quality crystals and the amount of time required to obtain visible crystals depended greatly on the robots used. The combined use of different robots, especially for protein samples exhibiting low crystallization success rates, significantly increased the chance of obtaining diffraction-quality crystals.

It was essential for the structural genomics of Thermus thermophilus HB8 to efficiently crystallize a number of proteins. To this end, three conventional robots, an HTS-80 (sitting-drop vapour diffusion), a Crystal Finder (hanging-drop vapour diffusion) and a TERA (modified microbatch) robot, were subjected to a crystallization condition screening test involving 18 proteins from T. thermophilus HB8. In addition, a TOPAZ (microfluidic free-interface diffusion) designed specifically for initial screening was also briefly examined. The number of diffraction-quality crystals and the time of appearance of crystals increased in the order HTS-80, Crystal Finder, TERA. With the HTS-80 and Crystal Finder, the time of appearance was short and the rate of salt crystallization was low. With the TERA, the number of diffraction-quality crystals was high, while the time of appearance was long and the rate of salt crystallization was relatively high. For the protein samples exhibiting low crystallization success rates, there were few crystallization conditions that were common to the robots used. In some cases, the success rate depended greatly on the robot used. The TOPAZ showed the shortest time of appearance and the highest success rate, although the crystals obtained were too small for diffraction studies. These results showed that the combined use of different robots significantly increases the chance of obtaining crystals, especially for proteins exhibiting low crystallization success rates. The structures of 360 of 944 purified proteins have been successfully determined through the combined use of an HTS-80 and a TERA.

The crystal structure of the complex of mouse Keap1-DC with a fragment of the nuclear protein prothymosin α was determined and refined to 1.9 Å resolution and revealed that the peptide binds to the bottom region of the β-propeller domain of Keap1-DC.

The Nrf2 transcription factor, which plays important roles in oxidative and xenobiotic stress, is negatively regulated by the cytoplasmic repressor Keap1. The β-propeller/Kelch domain of Keap1, which is formed by the double-glycine repeat and C-terminal region domains (Keap1-DC), interacts directly with the Neh2 domain of Nrf2. The nuclear oncoprotein prothymosin α (ProTα) also interacts directly with Keap1 and may play a role in the dissociation of the Keap1–Nrf2 complex. The structure of Keap1-DC complexed with a ProTα peptide (amino acids 39–54) has been determined at 1.9 Å resolution. The Keap1-bound ProTα peptide possesses a hairpin conformation and binds to the Keap1 protein at the bottom region of the β-propeller domain. Complex formation occurs as a consequence of their complementary electrostatic interactions. A comparison of the present structure with recently reported Keap1-DC complex structures revealed that the DLG and ETGE motifs of the Neh2 domain of Nrf2 and the ProTα peptide bind to Keap1 in a similar manner but with different binding potencies.

The ribosomal protein (L30e) from M. jannaschii was cloned from the gene MJ1044, expressed, purified and crystallized. The crystal belongs to the primitive tetragonal space group P43 and diffracted to 1.9 Å resolution.

In view of the biological significance of understanding the ribosomal machinery of both prokaryotes and eukaryotes, the L30e ribosomal protein from Methanocaldococcus jannaschii was cloned, overexpressed, purified and crystallized using the microbatch-under-oil method with the crystallization conditions 40% PEG 400, 0.1 M MES pH 6.0 and 5% PEG 3000 at 291 K. A diffraction-quality crystal (0.20 × 0.20 × 0.35 mm) was obtained that belonged to the primitive tetragonal space group P43, with unit-cell parameters a = 46.1, b = 46.1, c = 98.5 Å, and diffracted to a resolution of 1.9 Å. Preliminary calculations reveal that the asymmetric unit contains two monomers with a Matthews coefficient (V M) of 2.16 Å3 Da−1.

The crystal structure of the minimized α/β-hydrolase fold protein encoded by the gene TTHA1544 from T. thermophilus HB8 has been determined at 2.0 Å resolution.

The gene encoding TTHA1544 is a singleton found in the Thermus thermophilus HB8 genome and encodes a 131-amino-acid protein. The crystal structure of TTHA1544 has been determined at 2.0 Å resolution by the single-wavelength anomalous dispersion method in order to elucidate its function. There are two molecules in the asymmetric unit. Each molecule consists of four α-helices and six β-strands, with the β-strands composing a central β-sheet. A structural homology search revealed that the overall structure of TTHA1544 resembles the α/β-hydrolase fold, although TTHA1544 lacks the catalytic residues of a hydrolase. These results suggest that TTHA1544 represents the minimized α/β-hydrolase fold and that an additional component would be required for its activity.

A truncated variant of human ribosomal protien L10 was prepared and crystallized. Diffraction data were collected to 2.5 Å resolution.

Eukaryotic ribosomal protein L10 is an essential component of the large ribosomal subunit, which organizes the architecture of the aminoacyl-tRNA binding site. The human L10 protein is also called the QM protein and consists of 214 amino-acid residues. For crystallization, the L10 core domain (L10CD, Phe34–Glu182) was recombinantly expressed in Escherichia coli and purified to homogeneity. A hexagonal crystal of L10CD was obtained by the sitting-drop vapour-diffusion method. The L10CD crystal diffracted to 2.5 Å resolution and belongs to space group P3121 or P3221.

A putative member of the Lrp/AsnC family of transcriptional regulators, ST1022 from S. tokodaii strain 7, has been purified and crystallized in the absence and presence of the effector l-glutamine. A molecular-replacement solution was found using the FL11 transcriptional regulator from Pyrococcus sp. OT3 as a model and structural refinement is under way.

The Lrp/AsnC family of transcriptional regulators, also known as feast/famine transcriptional regulators, are widely distributed among bacteria and archaea. This family of proteins are likely to be involved in cellular metabolism, with exogenous amino acids functioning as effectors. Here, the crystallization and preliminary X-ray diffraction analysis of ST1022, a member of the Lrp/AsnC family of proteins, is reported with and without exogenous glutamine as the effector molecule. The crystals of native ST1022 and of the putative complex belong to the tetragonal space group I422, with unit-cell parameters a = b = 103.771, c = 73.297 Å and a = b = 103.846, c = 73.992 Å, respectively. Preliminary X-ray diffraction data analysis and molecular-replacement solution revealed the presence of one monomer per asymmetric unit.

The crystal structure of T. maritima tRNase Z has been determined at 1.97 Å. The structure shows the flexible arm and the fully metal-binding active site.

tRNA 3′-processing endoribonuclease (tRNase Z) is one of the enzymes involved in the 3′-end processing of precursor tRNAs and is a member of the metallo-β-lactamase superfamily. tRNase Z crystal structures have revealed that the enzyme forms a dimer and has a characteristic domain, named a flexible arm or an exosite, which protrudes from the metallo-β-lactamase core and is involved in tRNA binding. The refined structure of Thermotoga maritima tRNase Z has been determined at 1.97 Å resolution, revealing the structure of the flexible arm and the zinc-bound active site. The structure of the flexible arm of T. maritima tRNase Z is distinct from those of the Bacillus subtilis and Escherichia coli tRNase Zs. A comparison of the three tRNase Z structures revealed differences in the dimer orientation, which may be related to the unique cleavage-site specificity of T. maritima tRNase Z.

The C-terminal bromodomain of human BRD2 was cloned, expressed, purified and crystallized. A complete diffraction data set has been collected to 1.80 Å resolution.

BRD2 is a bromodomain-containing BET-family protein that associates with acetylated histones throughout the cell cycle. Although the tertiary structures of the bromodomains involved in histone acetyl transfer are already known, the structures of the BET-type bromodomains, which are required for tight association with acetylated chromatin, are poorly understood. Here, the expression, purification and crystallization of the C-terminal bromodomain of human BRD2 are reported. The protein was crystallized by the sitting-drop vapour-diffusion method in the orthorhombic space group P21212, with unit-cell parameters a = 71.78, b = 52.60, c = 32.06 Å and one molecule per asymmetric unit. The crystal diffracted beyond 1.80 Å resolution using synchrotron radiation.

A crystal structure of NADP+-bound T. thermophilus Δ1-pyrroline-5-carboxylate dehydrogenase refined to 1.55 Å resolution is reported. The structure provides structural insights into the mechanism of preference for coenzymes and enzyme activity.

Δ1-Pyrroline-5-carboxylate dehydrogenase (P5CDh) is known to preferentially use NAD+ as a coenzyme. The k cat value of Thermus thermophilus P5CDh (TtP5CDh) is four times lower for NADP+ than for NAD+. The crystal structure of NADP+-bound TtP5CDh was solved in order to study the structure–activity relationships for the coenzymes. The binding mode of NADP+ is essentially identical to that in the previously solved NAD+-bound form, except for the regions around the additional 2′-phosphate group of NADP+. The coenzyme-binding site can only accommodate this group by the rotation of a glutamate residue and subtle shifts in the main chain. The 2′-phosphate of NADP+ increases the number of hydrogen bonds between TtP5CDh and NADP+ compared with that between TtP5CDh and NAD+. Furthermore, the phosphate of the bound NADP+ would restrict the ‘bending’ of the coenzyme because of steric hindrance. Such bending is important for dissociation of the coenzymes. These results provide a plausible explanation of the lower turnover rate of NADP+ compared with NAD+.