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Endonuclease IV (EndoIV) is an endonuclease that acts at apurinic/apyrimidinic (AP) sites and is classified as either long-type or short-type. The crystal structures of representative types of EndoIV from Geobacillus kaustophilus and Thermus thermophilus HB8 were determined using X-ray crystallography. G. kaustophilus EndoIV (the long type) had a higher affinity for double-stranded DNA containing an AP-site analogue than T. thermophilus EndoIV (the short type). Structural analysis of the two different EndoIVs suggested that a C-terminal DNA-recognition loop that is only present in the long type contributes to its high affinity for AP sites. A mutation analysis showed that Lys267 in the C-terminal DNA-recognition loop plays an important role in DNA binding.

The Sulfolobus tokodaii protein ST0929 shares close structural homology with S. acidocaldarius maltooligosyl trehalose synthase (SaMTSase), suggesting that the two enzymes share a common enzymatic mechanism. MTSase is one of a pair of enzymes that catalyze trehalose biosynthesis. The relative geometries of the ST0929 and SaMTSase active sites were found to be essentially identical. ST0929 also includes the unique tyrosine cluster that encloses the reducing-end glucose subunit in Sulfolobus sp. MTSases. The current structure provides insight into the structural basis of the increase in the hydrolase side reaction that is observed for mutants in which a phenylalanine residue is replaced by a tyrosine residue in the subsite +1 tyrosine cluster of Sulfolobus sp.

Glyoxylate reductase catalyzes the NAD(P)H-linked reduction of glyoxylate to glycolate. Here, the 1.7 A crystal structure of glyoxylate reductase from the hyperthermophilic archaeon Pyrococcus horikoshii OT3 complexed with nicotinamide adenine dinucleotide phosphate [NADP(H)] determined by the single-wavelength anomalous dispersion (SAD) method is reported. The monomeric structure comprises the two domains typical of NAD(P)-dependent dehydrogenases: the substrate-binding domain (SBD) and the nucleotide-binding domain (NBD). The crystal structure and analytical ultracentrifugation results revealed dimer formation. In the NADP(H)-binding site, the pyrophosphate moiety and the 2'-phosphoadenosine moiety are recognized by the glycine-rich loop (residues 157-162) and by loop residues 180-182, respectively. Furthermore, the present study revealed that P. horikoshii glyoxylate reductase contains aromatic clusters and has a larger number of ion pairs and a lower percentage of hydrophobic accessible surface area than its mesophilic homologues, suggesting its thermostability mechanism.

Deoxyribonucleoside triphosphate triphosphohydrolase from Thermus thermophilus (Tt-dNTPase) has a unique regulatory mechanism for the degradation of deoxyribonucleoside triphosphates (dNTPs). Whereas the Escherichia coli homologue specifically hydrolyzes dGTP alone, dNTPs act as both substrate and activator for Tt-dNTPase. Here, the crystal structure of Tt-dNTPase has been determined at 2.2 A resolution, representing the first report of the tertiary structure of a dNTPase homologue belonging to the HD superfamily, a diverse group of metal-dependent phosphohydrolases that includes a variety of uncharacterized proteins. This enzyme forms a homohexamer as a double ring of trimers. The subunit is composed of 19 alpha-helices; the inner six helices include the region annotated as the catalytic domain of the HD superfamily. Structural comparison with other HD-superfamily proteins indicates that a pocket at the centre of the inner six helices, formed from highly conserved charged residues clustered around a bound magnesium ion, constitutes the catalytic site. Tt-dNTPase also hydrolyzed noncanonical dNTPs, but hardly hydrolyzed dNDP and dNMP. The broad substrate specificity for different dNTPs might be rationalized by the involvement of a flexible loop during molecular recognition of the base moiety. Recognition of the triphosphate moiety crucial for the activity might be attained by highly conserved positively charged residues. The possible mode of dNTP binding is discussed in light of the structure.

Nucleoside diphosphate (NDP) kinase contributes to the maintenance of cellular pools of all nucleoside triphosphates (NTPs) by catalyzing the transfer of the gamma-phosphoryl group from an NTP donor to an NDP acceptor. NDP kinase from the extreme thermophilic bacterium Thermus thermophilus HB8 was overexpressed in Escherichia coli and crystallized at 297 K using polyethylene glycol 8000 as the precipitant by means of the hanging-drop vapour-diffusion procedure. The crystals belong to the hexagonal system, space group P6(3)22, with unit-cell parameters a = b = 124.0, c = 104.9 A, alpha = beta = 90, gamma = 120 degrees. Assuming the asymmetric unit to contain two subunits, the calculated V(M) value was 3.7 A(3) Da(-1). The crystals diffracted X-rays generated by the synchrotron at SPring-8 to at least 1.9 A resolution and were suitable for high-resolution X-ray crystal structure determination.

An ADP-ribose pyrophosphatase from Thermus thermophilus HB8 was overproduced in Escherichia coli and purified. Gel-filtration chromatography showed the protein to be in a dimeric state. This protein catalyses the Mg(2+)- or Zn(2+)-dependent hydrolysis of ADP-ribose to AMP and ribose-5'-phosphate. It was crystallized in the absence and the presence of ADP-ribose by the hanging-drop vapour-diffusion method. Complete data sets were collected to 1.50 A resolution from the apo form using synchrotron radiation and to 2.0 A resolution from the complexed form. Both crystals belong to space group P3(1)21 or P3(2)21 and contain one molecule in the asymmetric unit.

The glycine-cleavage system is a multi-enzyme complex consisting of four different components (the P-, H-, T- and L-proteins). Recombinant H-protein corresponding to that from Thermus thermophilus HB8 has been overexpressed, purified and crystallized. Synchrotron radiation from BL44B2 at SPring-8 was used to collect a native data set to 2.5 A resolution. The crystals belonged to the hexagonal space group P6(5) and contained three molecules per asymmetric unit, with a solvent content of 39%. Because of the large number of molecules within a closely packed unit cell, this structure was solved by six-dimensional molecular replacement with the program EPMR using the pea H-protein structure as a search model and was refined to an R factor of 0.189 and a free R factor of 0.256. Comparison with the pea H-protein reveals two highly conserved regions surrounding the lipoyl-lysine arm. Both of these regions are negatively charged and each has additional properties that are conserved in H-proteins from many species, suggesting that these regions are involved in intermolecular interactions. One region has previously been proposed to constitute an interaction surface with T-protein, while the other may be involved in an interaction with P-protein. Meanwhile, the lipoyl-lysine arm of the T. thermophilus H-protein was found to be more flexible than that of the pea H-protein, supporting the hypothesis that H-protein does not form a stable complex with L-protein during the reaction.

Thermus thermophilus (Tth) HB8 glycine decarboxylase (P-protein) is an alpha(2)beta(2) tetrameric enzyme with a total molecular mass of 200 kDa. The alpha- and beta-subunits of the Tth P-protein have been coexpressed in Escherichia coli and purified as a stable complex. Dynamic light-scattering measurements indicated the recombinant protein to be monodisperse and its size to be consistent with an alpha(2)beta(2) tetrameric composition. Crystals of the protein have been grown in polyethylene glycol 3350 using the vapour-diffusion method at 291 K. Synchrotron radiation from BL45XU at SPring-8 was used to measure a complete native data set to 2.4 A resolution. The crystals belong to the trigonal space group P3(1)21 or P3(2)21, with unit-cell parameters a = b = 89.5, c = 371.0 A. Estimation of the crystal packing (V(M) = 2.15 A(3) Da(-1)) and self-rotation function analysis suggest the presence of one alpha(2)beta(2) tetramer per asymmetric unit, with the molecules related by non-crystallographic twofold symmetry.

A probable quinone oxidoreductase (MW = 32.1 kDa) from Thermus thermophilus HB8 was overproduced in Escherichia coli and purified. Gel-filtration chromatography suggested the protein to be in a dimeric state. This protein enhanced the reduction activity of quinones by NADPH. It was crystallized in the absence and the presence of NADPH by the hanging-drop vapour-diffusion method. Both crystals were hexagonal, space group P6(1)22 or P6(5)22, with unit-cell parameters a = b = 77.6, c = 236.7 A for the apo form and a = b = 77.6, c = 235.9 A for the complex with NADPH. They diffract to better than 2.3 A resolution with synchrotron radiation. The asymmetric unit has one protein subunit (V(M) = 3.2 A(3) Da(-1) and V(sol) = 0.62 for the apo form), indicating that the twofold axis of the dimeric protein and the crystallographic twofold axis coincide.

A DNA excision repair enzyme, UvrB, from Thermus thermophilus HB8 was crystallized by the vapor-diffusion method using lithium sulfate as the precipitant and beta-octylglucoside as an additive. The crystals belong to the trigonal space group P3121 or P3221, with unit-cell dimensions of a = b = 136.0 and c = 108.1 A. The crystal is most likely to contain one UvrB protein in an asymmetric unit with the Vm value of 3.8 A3 Da-1. The crystals diffracted X-rays beyond 2.9 A resolution. Although the crystals were sensitive to X-ray irradiation at room temperature, the frozen crystals at 100 K showed no apparent decay during the intensity measurement.